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Naegleria fowleri is a free-living amoeba which causes primary amoebic meningoencephalitis (PAM). Although PAM is rare, the fatality rate is staggering at over 97%. So, the importance of finding an effective treatment and cure for PAM caused by N. fowleri is a crucial area of research. Existing research on developing novel therapeutic strategies to counter N. fowleri infection is limited. Since the blood-brain barrier (BBB) presents an obstacle to delivering drugs to the site of infection, it is important to employ strategies that can effectively direct the therapeutics to the brain. In this regard, our review focuses on understanding the physiology and mechanisms by which molecules pass through the BBB, the current treatment options available for PAM, and the recent research conducted in the decade of 2012 to 2022 on the use of nanomaterials to enhance drug delivery. In addition, we compile research findings from other central nervous system (CNS) diseases that use shuttle peptides which allow for transport of molecules through the BBB. The approach of utilizing BBB shuttles to administer drugs through the BBB may open up new areas of drug discovery research in the field of N. fowleri infection.

Vermamoeba vermiformis (V.vermiformis) is one of the most common free-living amoeba (FLA) and is frequently found in environments such as natural freshwater areas, surface waters, soil, and biofilms. V. vermiformis has been reported as a pathogen with pathogenic potential for humans and animals. The aim is to report a case of non-Acanthamoeba keratitis in which V. vermiformis was the etiological agent, identified by culture and molecular techniques. Our case was a 48-year-old male patient with a history of trauma to his eye 10 days ago. The patient complained of eye redness and purulent discharge. A slit-lamp examination of the eye revealed a central corneal ulcer with peripheral infiltration extending into the deep stroma. The corneal scraping sample taken from the patient was cultured on a non-nutritious agar plate (NNA). Amoebae were evaluated according to morphological evaluation criteria. It was investigated by PCR method and confirmed by DNA sequence analysis. Although no bacterial or fungal growth was detected in the routine microbiological evaluation of the corneal scraping sample that was cultured, amoeba growth was detected positively in the NNA culture. Meanwhile, Acanthamoeba was detected negative by real-time PCR. However, V. vermiformis was detected positive with the specific PCR assay. It was confirmed by DNA sequence analysis to be considered an etiological pathogenic agent. Thus, topical administration of chlorhexidine gluconate %0.02 (8 × 1) was initiated. Clinical regression was observed 72 h after chlorhexidine initiation, and complete resolution of keratitis with residual scarring was noticed in 5 weeks. In conclusion, corneal infections due to free-living amoebae can occur, especially in poor hygiene. Although Acanthamoeba is the most common keratitis due to amoeba, V. vermiformis is also assumed to associate keratitis in humans. Clinicians should also be aware of other amoebic agents, such as V. vermiformis, in keratitis patients. [ABSTRACT FROM AUTHOR]

Balamuthia mandrillaris is a free-living amoeba (FLA) that causes granulomatous amebic encephalitis (GAE) and skin lesions. Transcriptomic analysis is a powerful tool used to study B. mandrillaris pathogenic infections. However, preliminary tests of RNA extraction showed poor results, so it has become essential to standardize a protocol for high-quality RNA. The present study evaluated 11 RNA extraction protocols based on three commercial kits by making modifications to the temperature and centrifugation times, and by combining kits. Four protocols, namely Q3 (based on QIAGEN RNeasy Mini Kit, with modifications in temperature and centrifugation times), T1 (Invitrogen TRIzol Reagent), T2 (combination of TRIzol and QIAGEN modified protocols) and T3 (combination of TRIzol and PROMEGA SV Total RNA Isolation protocols), presented RNA with good integrity and purity, except for the T1 protocol, which obtained an A260/230 value below the acceptable threshold. High RNA integrity (RIN) values were obtained with the Q3 (9.8), T2 (9.2), and T3 (8.9) protocols, while the T1 protocol obtained a lower RIN value (7.1). The Q3, T2, and T3 protocols obtained high-quality RNA from B. mandrillaris based on the criteria of integrity, purity, and concentration, where the implemented modifications and combinations raised the quality; thus, their use is recommended to obtain accurate results when performing transcriptomic analysis.

Abstract Naegleria fowleri, also known as brain-earing amoeba, causes severe and rapidly fatal CNS infection in humans called primary amebic meningoencephalitis (PAM). The DNA from the N. fowleri clinical isolate was sequenced for circular extrachromosomal ribosomal DNA (CERE - rDNA). The CERE contains 18 S, 5.8 S, and 28 S ribosomal subunits separated by internal transcribed spacers, 5 open reading frames (ORFs), and mostly repeat elements comprising 7268 bp out of 15,786 bp (46%). A wide variety of variations and recombination events were observed. Finally, the ORFs that comprised only 4 hypothetical proteins were modeled and screened against Zinc drug-like compounds. Two compounds [ZINC77564275 (ethyl 2-(((4-isopropyl-4 H-1,2,4-triazol-3-yl) methyl) (methyl)amino) oxazole-4-carboxylate) and ZINC15022129 (5-(2-methoxyphenoxy)-[2,2’-bipyrimidine]-4,6(1 H,5 H)-dione)] were finalized as potential druggable compounds based on ADME toxicity analysis. We propose that the compounds showing the least toxicity would be potential drug candidates after laboratory experimental validation is performed.

Balamuthia mandrillaris is a free-living amoeba (FLA) that causes granulomatous amebic encephalitis (GAE) and skin lesions. Transcriptomic analysis is a powerful tool used to study B. mandrillaris pathogenic infections. However, preliminary tests of RNA extraction showed poor results, so it has become essential to standardize a protocol for high-quality RNA. The present study evaluated 11 RNA extraction protocols based on three commercial kits by making modifications to the temperature and centrifugation times, and by combining kits. Four protocols, namely Q3 (based on QIAGEN RNeasy Mini Kit, with modifications in temperature and centrifugation times), T1 (Invitrogen TRIzol Reagent), T2 (combination of TRIzol and QIAGEN modified protocols) and T3 (combination of TRIzol and PROMEGA SV Total RNA Isolation protocols), presented RNA with good integrity and purity, except for the T1 protocol, which obtained an A260/230 value below the acceptable threshold. High RNA integrity (RIN) values were obtained with the Q3 (9.8), T2 (9.2), and T3 (8.9) protocols, while the T1 protocol obtained a lower RIN value (7.1). The Q3, T2, and T3 protocols obtained high-quality RNA from B. mandrillaris based on the criteria of integrity, purity, and concentration, where the implemented modifications and combinations raised the quality; thus, their use is recommended to obtain accurate results when performing transcriptomic analysis. [ABSTRACT FROM AUTHOR]

Naegleria fowleri, also known as brain-earing amoeba, causes severe and rapidly fatal CNS infection in humans called primary amebic meningoencephalitis (PAM). The DNA from the N. fowleri clinical isolate was sequenced for circular extrachromosomal ribosomal DNA (CERE - rDNA). The CERE contains 18 S, 5.8 S, and 28 S ribosomal subunits separated by internal transcribed spacers, 5 open reading frames (ORFs), and mostly repeat elements comprising 7268 bp out of 15,786 bp (46%). A wide variety of variations and recombination events were observed. Finally, the ORFs that comprised only 4 hypothetical proteins were modeled and screened against Zinc drug-like compounds. Two compounds [ZINC77564275 (ethyl 2-(((4-isopropyl-4 H-1,2,4-triazol-3-yl) methyl) (methyl)amino) oxazole-4-carboxylate) and ZINC15022129 (5-(2-methoxyphenoxy)-[2,2'-bipyrimidine]-4,6(1 H,5 H)-dione)] were finalized as potential druggable compounds based on ADME toxicity analysis. We propose that the compounds showing the least toxicity would be potential drug candidates after laboratory experimental validation is performed. [ABSTRACT FROM AUTHOR]

Mycobacterium leprae is an acid-fast bacterium that causes leprosy diseases, which remains a problem worldwide. Even though leprosy prevalence in the world has decreased significantly, many endemic pocket areas continue reporting new cases and harbor M. leprae in the environment, including water and soil. The presence of obligate intracellular bacteria-M. leprae in the environment raises a question on how it survives. Free-living amoeba has been proposed as its reservoir host in the environment. The study was conducted to give evidence that M. leprae can survive inside free-living amoeba isolated from water sources of leprosy endemic areas. M. leprae from leprosy patients was cultured together with Acanthamoeba sp. isolated from the water source of the leprosy endemic area. Viability and duplication of M. leprae inside amoeba then observed at day 14 and 28 using reverse transcriptase PCR and qPCR. The results showed that M. leprae survived inside the amoeba until day 28, but no bacterial replication was observed. The study reveals in vitro evidence of viable M. leprae inside free-living amoeba of leprosy endemic area environment. [ABSTRACT FROM AUTHOR]

Free-living amoebae (FLA) are widely distributed protozoa in both natural and artificial environments such as drinking water. In addition to the ability of all FLA to transport various pathogenic microorganisms, certain species, such as Acanthamoeba spp. or Balamuthia mandrillaris, have intrinsic pathogenic abilities and cause severe cerebral infections. Previous work has shown an enrichment of FLA cysts in biofilm developed in upper levels of Drinking Water Storage Towers (DWSTs), suggesting that differences in densities of FLA cysts may play a role in their unequal distribution in the water column. To evaluate this hypothesis, a model of a water column was created for this study and used to analyze the vertical distribution of cysts of the FLA Acanthamoeba castellanii, Vermamoeba vermiformis, and Balamuthia mandrillaris from 0 to 23 weeks. Interestingly, our data showed that the cysts of both A. castellanii and V. vermiformis were enriched in upper water levels during their aging. However, B. mandrillaris cysts were equally distributed in the water column during the entire study. These results show that, in addition to the role of water level variation in the DWST, some FLA cysts can become less dense during their aging, which contributes to their enrichment in upper water and therefore biofilm levels. [ABSTRACT FROM AUTHOR]

Rivers are important freshwater sources. They provide water for domestic and recreational use and serve as ecological sites for various microorganisms. Free-living amoebae are considered naturally occurring organisms in freshwater. However, their prevalence can suggest the water quality at a given source. Forty-five surface water samples from nine different sites were collected from the Marikina River. Samples were processed and cultured in non-nutrient agar (NNA) lawned with Escherichia coli, and were observed for 14 days using a light microscope. Thirty-six (80%) samples yielded a positive amoebic growth. Further molecular testing confirmed the presence of Acanthamoeba spp. using specific primer sets, JDP1 and JDP2. The current results of this study have shown a remarkable jump in the detection rate for free-living amoebae (FLAs). The continued deterioration of the Marikina River due to several human and non-human factors may have been the reason for this phenomenon. River clean-up, resettlement programs, and strict implementation of laws on waste disposal should be considered to rehabilitate the Marikina River. HIGHLIGHTS First report of isolation of Acanthamoeba spp. in the Marikina River.; Water quality assessment through FLA isolation.; First evaluation of health impacts of occurrence of FLAs.; First evaluation of impact on water quality of FLA occurrence.; First data on isolation of FLAs in a river.;

Naegleria fowleri is a free-living amoeba which causes primary amoebic meningoencephalitis (PAM). Although PAM is rare, the fatality rate is staggering at over 97%. So, the importance of finding an effective treatment and cure for PAM caused by N. fowleri is a crucial area of research. Existing research on developing novel therapeutic strategies to counter N. fowleri infection is limited. Since the blood–brain barrier (BBB) presents an obstacle to delivering drugs to the site of infection, it is important to employ strategies that can effectively direct the therapeutics to the brain. In this regard, our review focuses on understanding the physiology and mechanisms by which molecules pass through the BBB, the current treatment options available for PAM, and the recent research conducted in the decade of 2012 to 2022 on the use of nanomaterials to enhance drug delivery. In addition, we compile research findings from other central nervous system (CNS) diseases that use shuttle peptides which allow for transport of molecules through the BBB. The approach of utilizing BBB shuttles to administer drugs through the BBB may open up new areas of drug discovery research in the field of N. fowleri infection.

Traditional cysticidal assays for Acanthamoeba species revolve around treating cysts with compounds and manually observing the culture for evidence of excystation. This method is time-consuming, labor-intensive, and low throughput. We adapted and trained a YOLOv3 machine learning, object detection neural network to recognize Acanthamoeba castellanii trophozoites and cysts in microscopy images to develop an automated cysticidal assay. This trained neural network was used to count trophozoites in wells treated with compounds of interest to determine if a compound treatment was cysticidal. We validated this new assay with known cysticidal and noncysticidal compounds. In addition, we undertook a large-scale bioluminescence-based screen of 9,286 structurally unique marine microbial metabolite fractions against the trophozoites of A. castellanii and identified 29 trophocidal hits. These hits were then subjected to this machine learning-based automated cysticidal assay. One marine microbial metabolite fraction was identified as both trophocidal and cysticidal. IMPORTANCE The free-living Acanthamoeba can exist as a trophozoite or cyst and both stages can cause painful blinding keratitis. Infection recurrence occurs in approximately 10% of cases due to the lack of efficient drugs that can kill both trophozoites and cysts. Therefore, the discovery of therapeutics that are effective against both stages is a critical unmet need to avert blindness. Current efforts to identify new anti-Acanthamoeba compounds rely primarily upon assays that target the trophozoite stage of the parasite. We adapted and trained a machine learning, object detection neural network to recognize Acanthamoeba trophozoites and cysts in microscopy images. Our machine learning-based cysticidal assay improved throughput, demonstrated high specificity, and had an exquisite ability to identify noncysticidal compounds. We combined this cysticidal assay with our bioluminescence-based trophocidal assay to screen about 9,000 structurally unique marine microbial metabolites against A. castellanii. Our screen identified a marine metabolite that was both trophocidal and cysticidal.

Opportunistic premise plumbing pathogens (OPPPs) have been detected in buildings' plumbing systems causing waterborne disease outbreaks in the United States. In this study, we monitored the occurrence of OPPPs along with free-living amoeba (FLA) and investigated the effects of residential activities in a simulated home plumbing system (HPS). Water samples were collected from various locations in the HPS and analyzed for three major OPPPs: Legionella pneumophila, nontuberculous mycobacterial species (e.g., Mycobacterium avium, M. intracellulare, and M. abscessus), and Pseudomonas aeruginosa along with two groups of amoebas (Acanthamoeba and Vermamoeba vermiformis). A metagenomic approach was also used to further characterize the microbial communities. Results show that the microbial community is highly diverse with evidence of spatial and temporal structuring influenced by environmental conditions. L. pneumophila was the most prevalent pathogen (86% of samples), followed by M. intracellulare (66%) and P. aeruginosa (21%). Interestingly, M. avium and M. abscessus were not detected in any samples. The data revealed a relatively low prevalence of Acanthamoeba spp. (4%), while V. vermiformis was widely detected (81%) across all the sampling locations within the HPS. Locations with a high concentration of L. pneumophila and M. intracellulare coincided with the highest detection of V. vermiformis, suggesting the potential growth of both populations within FLA and additional protection in drinking water. After a period of stagnation lasting at least 2-weeks, the concentrations of OPPPs and amoeba immediately increased and then decreased gradually back to the baseline. Furthermore, monitoring the microbial population after drainage of the hot water tank and partial drainage of the entire HPS demonstrated no significant mitigation of the selected OPPPs. This study demonstrates that these organisms can adjust to their environment during such events and may survive in biofilms and/or grow within FLA, protecting them from stressors in the supplied water. [ABSTRACT FROM AUTHOR]

FLA-related conditions are a rare medical occurrence. Despite their rarity, they are considered a public health concern for two reasons: the absence of a regular treatment regimen in the case of central nervous system infections and the fast progression of the symptoms leading to fatal outcomes. A total of 358 articles were retrieved from different databases (91 from PubMed, 26 from NCBI, 138 from Academia, 102 from Science Direct, and one from IJMED). 7 (46.6%) clinical cases came from Egypt, 2 (13.3%) cases of FLA infection came from Nigeria, 3 (20%) cases came from the Gambia, and 1 (6.6%) case was reported from African countries like Algeria, Tunisia, South Africa, and Zambia. Medical conditions caused by free-living amoeba are considered significant public health concerns. These ubiquitous organisms can cause both fatal and debilitating health conditions. Immediate diagnosis of cases and proper hygienic practices are necessary to provide direct medical intervention. They may be the key to reducing the morbidity and mortality rates from FLA-acquired infections. Although several government-led initiatives have been implemented to mitigate a plethora of parasitic diseases, the case of FLA-related conditions in African countries has yet to be realized. [ABSTRACT FROM AUTHOR]

The aim of the study was to test the feasibility of long-term survival and preservation of the properties of Yersinia pestis in association with soil amoeba Acanthamoeba castellanii. Materials and methods. Y. pestis strains and acanthamoeba isolated in the common area of the Gorno-Altai high-mountain plague focus were used for the study. The systematic affiliation of protozoa was determined through analyzing the 18S rRNA gene fragment sequencing data, followed by alignment with amoeba sequences from the NCBI GenBank database. A fluorescent Y. pestis strain was obtained by electroporation using the pTurboGFP-B plasmid. Co-cultivation was carried out in saline buffer in the absence of nutrients for the cells of plague pathogen. The influence of co-culturing with protozoa on Y. pestis properties was determined using microbiological, biological, and molecular-genetic methods. Results and discussion. The cell viability preservation for 22 months of the experiment in Y. pestis strain belonging to the main subspecies of the antique biovar, the 4.ANT phylogenetic line in co-culture with amoeba cells in the absence of additional nutrients has been established. Co-cultivation with amoebae did not lead to a change in the cultural, morphological, genetic and virulent properties of the plague pathogen strain. The data obtained confirm the possibility of using Acanthamoeba castellanii by the plague microbe to persist in soil biocenoses and open up the prospect of studying the mechanisms of plague pathogen surviving during extended inter-epizootic periods.

Free-living amoebae (FLA) are widely distributed protozoa in both natural and artificial environments such as drinking water. In addition to the ability of all FLA to transport various pathogenic microorganisms, certain species, such as Acanthamoeba spp. or Balamuthia mandrillaris, have intrinsic pathogenic abilities and cause severe cerebral infections. Previous work has shown an enrichment of FLA cysts in biofilm developed in upper levels of Drinking Water Storage Towers (DWSTs), suggesting that differences in densities of FLA cysts may play a role in their unequal distribution in the water column. To evaluate this hypothesis, a model of a water column was created for this study and used to analyze the vertical distribution of cysts of the FLA Acanthamoeba castellanii, Vermamoeba vermiformis, and Balamuthia mandrillaris from 0 to 23 weeks. Interestingly, our data showed that the cysts of both A. castellanii and V. vermiformis were enriched in upper water levels during their aging. However, B. mandrillaris cysts were equally distributed in the water column during the entire study. These results show that, in addition to the role of water level variation in the DWST, some FLA cysts can become less dense during their aging, which contributes to their enrichment in upper water and therefore biofilm levels.

Opportunistic premise plumbing pathogens (OPPPs) have been detected in buildings’ plumbing systems causing waterborne disease outbreaks in the United States. In this study, we monitored the occurrence of OPPPs along with free-living amoeba (FLA) and investigated the effects of residential activities in a simulated home plumbing system (HPS). Water samples were collected from various locations in the HPS and analyzed for three major OPPPs: Legionella pneumophila, nontuberculous mycobacterial species (e.g., Mycobacterium avium, M. intracellulare, and M. abscessus), and Pseudomonas aeruginosa along with two groups of amoebas (Acanthamoeba and Vermamoeba vermiformis). A metagenomic approach was also used to further characterize the microbial communities. Results show that the microbial community is highly diverse with evidence of spatial and temporal structuring influenced by environmental conditions. L. pneumophila was the most prevalent pathogen (86% of samples), followed by M. intracellulare (66%) and P. aeruginosa (21%). Interestingly, M. avium and M. abscessus were not detected in any samples. The data revealed a relatively low prevalence of Acanthamoeba spp. (4%), while V. vermiformis was widely detected (81%) across all the sampling locations within the HPS. Locations with a high concentration of L. pneumophila and M. intracellulare coincided with the highest detection of V. vermiformis, suggesting the potential growth of both populations within FLA and additional protection in drinking water. After a period of stagnation lasting at least 2-weeks, the concentrations of OPPPs and amoeba immediately increased and then decreased gradually back to the baseline. Furthermore, monitoring the microbial population after drainage of the hot water tank and partial drainage of the entire HPS demonstrated no significant mitigation of the selected OPPPs. This study demonstrates that these organisms can adjust to their environment during such events and may survive in biofilms and/or grow within FLA, protecting them from stressors in the supplied water.

Introduction: Balamuthia (B.) mandrillaris is a free-living amoeba that can cause rare yet fatal granulomatous amoebic encephalitis (GAE). However, efficacious treatment for GAE is currently unavailable, especially when genomic studies on B. mandrillaris are limited. Methods: In this study, B. mandrillaris strain KM-20 was isolated from the brain tissue of a GAE patient, and its mitochondrial genome was de novo assembled using high-coverage Nanopore long reads and Illumina short reads. Results and Discussion: Phylogenetic and comparative analyses revealed a range of diversification in the mitochondrial genome of KM-20 and nine other B. mandrillaris strains. According to the mitochondrial genome alignment, one of the most variable regions was observed in the ribosomal protein S3 (rps3), which was caused by an array of novel protein tandem repeats. The repeating units in the rps3 protein tandem region present significant copy number variations (CNVs) among B. mandrillaris strains and suggest KM-20 as the most divergent strain for its highly variable sequence and highest copy number in rps3. Moreover, mitochondrial heteroplasmy was observed in strain V039, and two genotypes of rps3 are caused by the CNVs in the tandem repeats. Taken together, the copy number and sequence variations of the protein tandem repeats enable rps3 to be a perfect target for clinical genotyping assay for B. mandrillaris. The mitochondrial genome diversity of B. mandrillaris paves the way to investigate the phylogeny and diversification of pathogenic amoebae. [ABSTRACT FROM AUTHOR]

Les parasitoses et mycoses oculaires regroupent de nombreuses pathologies dont la sévérité varie selon la nature du parasite ou du champignon. Elles peuvent menacer la vue, aussi doivent-elles être diagnostiquées et traitées en urgence. Elles surviennent dans des circonstances différentes (traumatisme, greffe de cornée, contexte immunosuppresseur local ou général, séjour en zone tropicale, pratiques traditionnelles particulières). L'infection peut être isolée ou intégrée dans une infection systémique. Le diagnostic clinique de ces infections est souvent difficile car les lésions peuvent ne pas être spécifiques à l'agent pathogène. Par conséquent, le diagnostic biologique est primordial pour permettre une prise en charge adéquate. Celui-ci est réalisé selon différentes stratégies en fonction des lésions et des agents infectieux suspectés. Il nécessite une combinaison de différents tests microbiologiques (examen direct, culture, PCR, sérologie) et doit tenir compte de la difficulté, du risque invasif et des faibles volumes des prélèvements oculaires. Le recours à des laboratoires spécialisés (mycologie-parasitologie), voire experts, est ainsi inévitable. Ocular parasitosis and mycosis include numerous pathologies whose severity varies according to the nature of the parasite or fungus. They can threaten the sight, so they must be diagnosed and treated in emergency. They occur in different circumstances (trauma, corneal transplantation, local or general immunosuppressive context, stay in tropical area, particular traditional practices). The infection can be isolated or integrated in a systemic infection. The clinical diagnosis of these infections is often difficult because the lesions may not be specific to the pathogen. Therefore, biological diagnosis is essential for proper management. Biological diagnosis is performed according to different strategies depending on the lesions and the suspected infectious agents. It requires a combination of different microbiological tests (direct examination, culture, PCR, serology) and must take into account the difficulty, invasive risk and small volumes of ocular samples. The use of specialized laboratories (mycology-parasitology), or even experts, is thus inevitable. [ABSTRACT FROM AUTHOR]

Free-living amoeba Naegleria fowleri causes a rapidly fatal infection primary amebic meningoencephalitis (PAM) in children. The drug of choice in treating PAM is amphotericin B, but very few patients treated with amphotericin B have survived PAM. Therefore, development of efficient drugs is a critical unmet need. We identified that the FDA-approved pitavastatin, an inhibitor of HMG Co-A reductase involved in the mevalonate pathway, was equipotent to amphotericin B against N. fowleri trophozoites. The genome of N. fowleri contains a gene encoding protein farnesyltransferase (FT), the last common enzyme for products derived from the mevalonate pathway. Here, we show that a clinically advanced FT inhibitor lonafarnib is active against different strains of N. fowleri with EC50 ranging from 1.5 to 9.2 µM. A combination of lonafarnib and pitavastatin at different ratios led to 95% growth inhibition of trophozoites and the combination achieved a dose reduction of about 2- to 28-fold for lonafarnib and 5- to 30-fold for pitavastatin. No trophozoite with normal morphology was found when trophozoites were treated for 48 h with a combination of 1.7 µM each of lonafarnib and pitavastatin. Combination of lonafarnib and pitavastatin may contribute to the development of a new drug regimen for the treatment of PAM.

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. HIGHLIGHTS First report of Acanthamoeba astronyxis in groundwater sources in the Philippines.; First report of Acanthamoeba genotype T4 in groundwater sources in the Philippines.; Confirmed pathogenic Acanthamoeba spp. through thermo-tolerance testing in groundwater.; First extensive survey of FLAs in groundwater sources in the Philippines.; Potential risks of FLA occurrence in groundwater sources.;

IntroductionBalamuthia (B.) mandrillaris is a free-living amoeba that can cause rare yet fatal granulomatous amoebic encephalitis (GAE). However, efficacious treatment for GAE is currently unavailable, especially when genomic studies on B. mandrillaris are limited.MethodsIn this study, B. mandrillaris strain KM-20 was isolated from the brain tissue of a GAE patient, and its mitochondrial genome was de novo assembled using high-coverage Nanopore long reads and Illumina short reads.Results and DiscussionPhylogenetic and comparative analyses revealed a range of diversification in the mitochondrial genome of KM-20 and nine other B. mandrillaris strains. According to the mitochondrial genome alignment, one of the most variable regions was observed in the ribosomal protein S3 (rps3), which was caused by an array of novel protein tandem repeats. The repeating units in the rps3 protein tandem region present significant copy number variations (CNVs) among B. mandrillaris strains and suggest KM-20 as the most divergent strain for its highly variable sequence and highest copy number in rps3. Moreover, mitochondrial heteroplasmy was observed in strain V039, and two genotypes of rps3 are caused by the CNVs in the tandem repeats. Taken together, the copy number and sequence variations of the protein tandem repeats enable rps3 to be a perfect target for clinical genotyping assay for B. mandrillaris. The mitochondrial genome diversity of B. mandrillaris paves the way to investigate the phylogeny and diversification of pathogenic amoebae.

Although the presence of free-living amoebae (FLAs) in various water sources has been reported, few studies have been surveyed on their abundance in medical-unit and dental-unit water samples. The current study aimed to identify morphological and molecular characteristics of FLA isolates in the water samples of the dental unit in Iran. A total of 232 water samples were collected from 17 dental units. Then, filtration and cultivation were conducted on a non-nutrient agar (NNA) medium. Also, polymerase chain reaction (PCR) assay and sequencing were performed by using the genus/species-specific primers plus a common primer set on positive samples. One hundred and sixty-six samples were positive for FLA by the microscopic method, whereas 114 samples were positive by the molecular method with a common primer set. Considering the PCR assay with genus/species-specific primers, 23.27% (54/232) samples were identified as Acanthamoeba spp. (belonging to T4 genotype), 36.63% (85/232) as Vermamoeba vermiformis, and 1.72% (4/232) as Vahlkampfiidae family (Naegleria lovaniensis). These results highlight a need to improve filtration systems in dental units and periodic screenings for FLA in dental-unit water. HIGHLIGHTS Among 232 water samples of the dental unit, 166 samples were positive for FLA by the microscopic method, whereas 114 samples were positive by the molecular method with a common primer set.; Considering the PCR, 23.27% (54/232) samples were identified as Acanthamoeba spp. (belonging to T4 genotype), 36.63% (85/232) as Vermamoeba vermiformis, and 1.72% (4/232) as Vahlkampfiidae family (Naegleria lovaniensis).;

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'-TCTCA-CAAGCTGCTAGGGAGTCA-3') primers, and universal primer Euk A (5'-AACCTGGTTGATCCTGCCAGT-3') and Euk B (5'-TGATCCTTCTGCA GGTTCACCTAC-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. [ABSTRACT FROM AUTHOR]

As part of the Eskom rebate programme, energy-efficient hot water systems such as solar water heaters (low pressure), heat pumps and energy-efficient showerheads were rolled out to the public as a measure to conserve and save energy. There has been a concern that these systems may not reach the required high temperatures, especially during winter, and, as a result of this, Acanthamoeba and its associated bacteria such as Legionella pneumophila, Pseudomonas aeruginosa and nontuberculous mycobacteria could flourish within these systems causing a potential health risk to consumers. This study examined the relationship between Acanthamoeba and its associated bacteria at different temperature ranges. A total of 156 water (69) and biofilm samples (87) were collected from a solar water heater, heat pump, geyser and showerheads and examined for these organisms using amoebal enrichment and molecular techniques. Amoeba could be cultivated from 45 (65.2%) of the water samples and 56 (64.4%) of the biofilm samples. The study confirmed the presence of Legionella pneumophila, Pseudomonas aeruginosa and nontuberculous mycobacteria in the hot water systems at both of the simulated winter (20°C to 30°C) and higher summer (40°C to 55°C) temperatures as well as the control system. There was a significant positive correlation between the presence of Acanthamoeba and the presence of Pseudomonas. Based on this association it is suggested that Pseudomonas aeruginosa could be investigated as an indicator organism for the presence of Acanthamoeba and opportunistic pathogens. [ABSTRACT FROM AUTHOR]

Simple Summary: Acanthamoeba is free-living amoeba known to cause severe vision threatening eye infection. In most of the cases treating this infection is difficult due to the Acanthamoeba's ability to switch between an active feeding stage (trophozoite) and inactive dormant stage (cyst). Arginine rich peptides are highly positively charged and can kill the microorganisms by acting on their negatively charged surface. This study assessed the anti-amoebic activity of polyhomoarginines of different lengths against Acanthamoeba castellanii trophozoites and cysts. Polyhomoarginine showed excellent anti-amoebic activity against both the stages of Acanthamoeba castellanii. Arginine-rich peptides can have broad-spectrum anti-bacterial and anti-fungal activities. Polyhomoarginine consists of highly cationic residues which can act on the negatively charged microbial cell membranes. Acanthamoeba is a free-living protozoan known to cause a rare corneal infection which is difficult to diagnose and treat. This study evaluated the activity of the polyhomoarginines against Acanthamoeba castellanii. Acanthamoeba amoebicidal, amoebistatic, encystation and excystment assays were performed using protocols described in the literature. The activity of polyhomoarginines (PHAs) of different lengths (10 to 400 residues) was measured against the trophozoites and cysts of Acanthamoeba castellanii ATCC30868 in concentrations ranging from 0.93 μM to 15 μM. Data were represented as mean ± SE and analysed using one-way ANOVA. Overall, PHAs demonstrated good anti-acanthamoeba activity against both trophozoites and cysts. PHA 30 reduced the number of viable trophozoites by 99%, inhibited the formation of cysts by 96% and the emergence of trophozoites from cysts by 67% at 3.75 μM. PHA 10 was similarly active, but at a slightly higher concentration of 15 μM, reducing the numbers of viable trophozoites by 98%, inhibiting cyst formation by 84% and preventing the emergence of trophozoites from cysts by 99%. At their greatest anti-amoeba concentrations, PHA 10 gave only 8% haemolysis at 15 μM while PHA 30 gave <40 % haemolysis at 3.75 μM. Polyhomoarginine 10 showed excellent anti-amoebic activity against both forms of Acanthamoeba castellanii and was non-toxic at its most active concentrations. This implies that polyhomoarginines can be developed into a potential therapeutic agent for Acanthamoeba keratitis. However, there is a need to carry out further pre-clinical and then in vivo experiments in the AK animal model. [ABSTRACT FROM AUTHOR]

Acanthamoeba is a free-living protozoan known to cause keratitis most commonly, especially among contact lens wearers. Treatment of Acanthamoeba keratitis is challenging as Acanthamoeba can encyst from the active form, a trophozoite, into a hibernating cyst that is refractory to antibiotics and difficult to kill; therefore, there is a need for more effective anti-amoebic strategies. In this study, we have evaluated the anti-amoebic activity of the antimicrobial peptide mimic RK-758 against Acanthamoeba castellanii. RK-758 peptidomimetic was subjected to biological assays to investigate its amoebicidal, amoebistatic, anti-encystation, and anti-excystation effects on A. castellanii. The anti-amoebic activity of the peptide mimic RK-758 was compared with chlorhexidine against the Acanthamoeba castellanii ATCC30868 and Acanthamoeba castellanii 044 (a clinical strain) with the concentrations of both ranging from 125 µM down to 7.81 µM. All experiments were performed in duplicate with three independent replicates. The data were represented as mean ± SE and analysed using a two-sample t-test and two-tailed distributions. A p < 0.05 was considered statistically significant. The peptidomimetic RK-758 had anti-Acanthamoeba activity against both trophozoites and cysts in a dose-dependent manner. The RK-758 had amoebicidal and growth inhibitory activities of ≥50% at a concentration between 125 µM and 15.6 µM against the trophozoites of both Acanthamoeba strains. Inhibitory effects on the cyst formation and trophozoite re-emergence from cysts were noted at similar concentrations. Chlorhexidine had 50% activity at 7.81 µM and above against the trophozoites and cysts of both strains. In the haemolysis assay, the RK-758 lysed horse RBCs at concentrations greater than 50 µM whereas lysis occurred at concentrations greater than 125 µM for the chlorhexidine. The peptidomimetic RK-758, therefore, has activity against both the trophozoite and cyst forms of Acanthamoeba and has the potential to be further developed as an anti-microbial agent against Acanthamoeba. RK-758 may also have use as an anti-amoebic disinfectant in contact lens solutions. [ABSTRACT FROM AUTHOR]

Acanthamoeba spp. are the most common free-living amoeba worldwide, inducing life-threatening diseases such as Granulomatous Amoebic Encephalitis, pulmonary infection, and amoebic keratitis. This study aimed to identify the FLA and Acanthamoeba genotypes in patients with pulmonary symptoms suspected of cancer in Kashan's hospitals, Kashan, Iran. This cross-sectional study was conducted on 97 bronchoalveolar lavage samples of patients with respiratory symptoms suspected of lung cancer, who were admitted to the Shahid Beheshti Hospital of Kashan from 2019 to 2020. The samples were cultured onto 1.5% non-nutrient agar enriched with killed Escherichia coli and examined for the presence of FLA. Following amoeba isolation and DNA extraction, Acanthamoeba spp. were determined by Polymerase Chain Reaction using JDP1 and JDP2 primers, which amplified a 490 bp fragment from the 18 S rDNA gene. Eighteen Acanthamoeba isolates were sequenced, and the genotypes were identified. The prevalence of FLA and Acanthamoeba and the relationship between symptoms and demographic variables were analyzed with SPSS Software version 16. The prevalence rates of FLA and Acanthamoeba in the BAL samples was 86.6% and 73.2%, respectively. All Acanthamoeba isolates belonged to the T4 genotype. The most symptoms among Acanthamoeba-positive patients were dyspnea and cough; however, their difference was not statistically significant. The findings indicated the high prevalence of FLA and Acanthamoeba in BAL in the population suspected of cancer in Kashan. Since the T4 genotype is a pathogenic genotype of Acanthamoeba, training health and improving sanitation levels would help to prevent infection. [ABSTRACT FROM AUTHOR]

Background: Pathogenic free-living amoeba (FLA) such as Acanthamoeba spp., Naegleria fowleri, and Balamuthia mandrillaris are causative agents of fatal amoebic encephalitis/meningoencephalitis. The diagnosis of such infections is challenging due to a lack of clinical suspicion and expertise in microscopic identification. We evaluated the performance of molecular assays for the timely and accurate detection of FLA-causing central nervous system (CNS) afflictions. Methods: This study included samples from 156 patients with suspected encephalitis/meningoencephalitis, including 149 cerebrospinal fluid (CSF) samples, 5 brain tissue biopsies, and 2 brain abscess samples. All the samples were subjected to PCR-based detection of Acanthamoeba spp., N. fowleri, and B. mandrillaris. The diagnostic characteristics and the inter-rater reliability scores were evaluated for parasite-specific polymerase chain reaction (PCR) using culture on non-nutrient agar (NNA)/microscopy or histopathological examination as a confirmatory test for Acanthamoeba spp. and N. fowleri and histopathology for B. mandrillaris. Results: We detected 11 samples positive for FLA, including 6 Acanthamoeba spp., 3 B. mandrillaris, and 2 N. fowleri. Furthermore, all 11 samples were positive according to the confirmatory tests, i.e., culture on NNA/microscopy/histopathology in the case of Acanthamoeba spp. and N. fowleri and histopathology of tissue biopsies for B. mandrillaris. The inter-rater reliability between the PCRs and the confirmatory tests for the detection of Acanthamoeba spp., N. fowleri, and B. mandrillaris was 100%. Conclusions: The PCR-based detection of FLA in patients suspected of encephalitis/meningoencephalitis was found to be fast, efficient, and reliable in our study. We suggest the use of these PCRs in laboratories to obtain additional data on their efficiency in diagnosing FLA infections of the CNS. The present study was conducted with a small sample size of 156 patient samples, and we found only six Acanthamoeba spp., three B. mandrillaris, and two N. fowleri. The present study should be conducted on a larger sample size for better evaluation of the primer pairs. [ABSTRACT FROM AUTHOR]

Free-living amoebas (FLAs) can cause neurological and ocular complications in humans. Water supplies play a critical role in transmitting FLAs to humans. The aim of the present study was to investigate the presence of FLAs in various aquatic sources including drinking water, stagnant water, and surface water in Alborz province, northern Iran, using morphological and molecular techniques. A total of 70 water samples were collected from 34 drinking waters, 23 surface waters, and 13 stagnant waters. Filtration and cultivation were employed to isolate FLAs. PCR assay was applied by using the genus-specific primers on positive samples. Pathogenicity tests (osmo- and thermo-tolerance properties) were performed for Acanthamoeba spp., positive sample. Considering the morphological criteria, four positive samples of Acanthamoeba sp., three Vermamoeba sp., two mixed Vermamoeba sp. with Vahlkamfiids, and one mixed Acanthamoeba sp. with Vahlkamfiids were isolated. Five Acanthamoeba sp. isolates were amplified using the JDP primer pairs. Among them, two genotypes, T4 (three isolates) and T5 (two isolates) corresponding to A. lenticulata, were identified. Four V. vermiformis samples were confirmed using the sequencing. This study highlighted the occurrence of potentially pathogenic waterborne FLAs in water habitats associated with high human activity. The results of such research on the prevalence of FLAs, as a human hazard, should be communicated to health policymakers. HIGHLIGHTS Water samples were collected from drinking, surface, and stagnant waters.; According to the morphological assessment, Acanthamoeba, Vermamoeba, and Vahlkamfiids were detected among samples.; The genotypes T4 and T5 (corresponding to A. lenticulata) were characterized.;

Naegleria. fowleri, a protozoa belonging to the free-living amoeba group, is the causative agent of a central nervous system affecting disease that is fatal in more than the 95% of the reported cases. This parasite can be found in warm water bodies such as lakes, rivers or inadequately disinfected swimming pools. On the other hand, chlorination and UV light treatment are two of the most extensively used disinfection methods in recreational water facilities.In this study the effect of chlorination and UV light on N. fowleri trophozoites was studied in a close water circuit with the aim to assess the efficacy of this disinfection methods in large pools. The obtained results showed that the chlorination was able to decrease the number of viable cells despite the elimination was not totally achieved. Nonetheless, the combination of the UV light with the chlorination allowed the complete removal of the N. fowleri trophozoites from the water in experimental testing conditions.

Free-living amoebae (FLA) are considered environmental pathogens and thus pose a public health threat. Their ubiquity in natural sources may magnify the potential severity of health outcomes in the future. However, less attention was given despite several probable public health risks that arise from the presence of pathogenic strains in the environment. Here, we provide epidemiological data based on investigations involving the distribution and occurrence of free-living amoebae in the Republic of the Philippines. This aims to connect data of fragmented studies of these organisms and provide potential roadmaps in FLA research in the country. The majority of the reviewed articles (n = 19) focused on characterization studies (36.8%; 7/19) while environmental isolation and isolation from biological samples had an equal frequency of 31.6% (6/19) each. There is a great disparity between the established ubiquity in environmental sources and the number of cases of FLA infections in the country. FLA-related research in the Philippines is still in its inceptive stage with several gaps to fill, which can be used to formulate policy briefs in the future regarding its isolation, identification, diagnosis, therapeutic management, and control of FLA infections in the country. [ABSTRACT FROM AUTHOR]

Naegleria fowleri ( N. fowleri ) infection via the upper respiratory tract causes a fatal CNS disease known as primary amoebic meningoencephalitis (PAM). The robust in vivo immune response to N. fowleri infection underlies the immunopathology that characterizes the disease. However, little is known about why this pathogen evades immune control. Infections occur in seemingly healthy individuals and effective clinical options are lacking, thus a nearly 98% fatality rate. It is unclear how or if host factors may contribute to susceptibility or disease exacerbation, yet mechanistic studies of the in vivo immune response and disease progression are hampered by a lack of tools. In this study, we have generated monoclonal antibodies to N. fowleri surface antigens and shown them to be excellent tools for studying the in vivo immune response. We also identified one monoclonal, 2B6, with potent inherent anti-amoebastatic activity in vitro . This antibody is also able to therapeutically prolong host survival in vivo and furthermore, recombinant antibodies with an isotype more capable of directing immune effector activity further improved survival when given therapeutically. Thus, we report the generation of a novel monoclonal antibody to N. fowleri that can enhance beneficial immune functions, even when given therapeutically during disease. We believe this provides evidence for the potential of therapeutic antibody treatments in PAM.IMPORTANCE Naegleria fowleri ( N. fowleri ) is a free-living amoeba that is found ubiquitously in warm freshwater. While human exposure is common, it rarely results in pathogenesis. However, when N. fowleri gains access to the upper airway, specifically the olfactory mucosa, infection leads to a lethal disease known as primary amoebic meningoencephalitis (PAM). As a free-living amoeba, N. fowleri does not need a mammalian host; indeed, it can be accurately described as an accidental opportunistic pathogen. While most opportunistic infections occur in humans who are immunocompromised, there are no reported immune dysfunctions associated with N. fowleri infection. Therefore, the basis for N. fowleri opportunism is not known, and the reasons why some humans develop PAM while others do not are simply not well understood. It is reasonable to speculate that local or acute immune failures, potentially even a lack of prior adaptive immunity, are related to disease susceptibility. Careful immune profiling and characterization of the in vivo immune response to N. fowleri in a mammalian host are desperately needed to understand which host factors are critical to defense, and how these responses might be compromised in a way that results in lethal infection. To identify genes and pathways that provide resistance against in vivo N. fowleri infection, we generated surface reactive monoclonal antibodies (Abs) that provide rapid amoeba detection and quantification in vivo . Interestingly, N. fowleri binding Abs have been readily detected in the serum and saliva of humans and animals suggesting that non-lethal exposure drives a humoral immune response against the amoeba. Yet, how Abs might interact with Naegleria in vivo or contribute to preventing lethal infection is not well understood. In this study, we have generated and characterized a monoclonal antibody (Ab), Clone 2B6, that recognizes a glycosylated surface antigen present in cultured in vitro N. fowleri as well as mouse passaged N. fowleri . When clone 2B6 binds to N. fowleri , it inhibits amoeba motility and feeding behavior, leading to strong growth inhibition. Mice treated systemically and intracerebrally with Ab displayed a delayed disease onset and prolonged survival. In addition, we found that enhancing immune-directed effector activity via antibody isotype could further enhance survival without obvious immunopathogenic side effects. These findings show the potential for antibody treatment as an additional therapeutic to those used currently in PAM., Competing Interests: The authors declare no conflict of interest.

Acanthamoeba castellanii is a widespread Free-Living Amoeba (FLA) that can cause severe ocular or cerebral infections in immunocompetent and immunocompromised patients, respectively, besides its capacity to transport diverse pathogens. During their life cycle, FLA can alternate between a vegetative form, called a trophozoite, and a latent and resistant form, called a cyst. This resistant form is characterized by the presence of a cell wall containing two layers, namely the ectocyst and the endocyst, mainly composed of cellulose and proteins. In the present work, we aimed to stimulate Acanthamoeba castellanii excystment by treating their cysts with a cellulolytic enzyme, i.e., cellulase, or two proteolytic enzymes, i.e., collagenase and pepsin. While 11 days were necessary to obtain total excystment in the control at 27°C, only 48 h were sufficient at the same temperature to obtain 100% trophozoites in the presence of 25 U/mL cellulase, 50 U/mL collagenase or 100 U/mL pepsin. Additionally, more than 96% amoebae have excysted after only 24 h with 7.5 U/mL cellulase at 30°C. Nevertheless, no effect of the three enzymes was observed on the excystment of Balamuthia mandrillaris and Vermamoeba vermiformis. Surprisingly, A. castellanii trophozoites excysted in the presence of cellulase displayed a markedly shorter doubling time at 7 h, in comparison to the control at 23 h. Likewise, trophozoites doubled their population in 9 h when both cellulose and cellulase were added to the medium, indicating that Acanthamoeba cyst wall degradation products promote their trophozoite proliferation. The analysis of cysts in epifluorescent microscopy using FITC-lectins and in electron microscopy revealed a disorganized endocyst and a reduction of the intercystic space area after cellulase treatment, implying that these cellular events are preliminary to trophozoite release during excystment. Further studies would be necessary to determine the signaling pathways involved during this amoebal differentiation process to identify new therapeutic targets for the development of anti-acanthamoebal drugs.

Although the presence of free-living amoebae (FLAs) in various water sources has been reported, few studies have been surveyed on their abundance in medical-unit and dental-unit water samples. The current study aimed to identify morphological and molecular characteristics of FLA isolates in the water samples of the dental unit in Iran. A total of 232 water samples were collected from 17 dental units. Then, filtration and cultivation were conducted on a non-nutrient agar (NNA) medium. Also, polymerase chain reaction (PCR) assay and sequencing were performed by using the genus/species-specific primers plus a common primer set on positive samples. One hundred and sixty-six samples were positive for FLA by the microscopic method, whereas 114 samples were positive by the molecular method with a common primer set. Considering the PCR assay with genus/species-specific primers, 23.27% (54/232) samples were identified as Acanthamoeba spp. (belonging to T4 genotype), 36.63% (85/232) as Vermamoeba vermiformis, and 1.72% (4/232) as Vahlkampfiidae family (Naegleria lovaniensis). These results highlight a need to improve filtration systems in dental units and periodic screenings for FLA in dental-unit water. [ABSTRACT FROM AUTHOR]

Background: Naegleria fowleri, the causative agent of primary amoebic meningoencephalitis (PAM), is a free-living amoeba. It is a water-borne infection usually detected in children and young people with healthy immune system who swim, dive and perform activities in fresh and hot springs. Purpose: In this study, it was aimed to raise awareness in the differential diagnosis of meningitis etiopathogenesis by showing that N. fowleri may also be the causative agent, albeit very rarely, in meningitis cases in Turkey. Methods: Our case was an 18-year-old male patient whose relatives stated that he has gone to the hot spring; his headache complaint started after 2 to 3 days after return from the hot spring. Cerebrospinal fluid (CSF) sample taken from the patient was investigated by direct microscopic examination, real-time PCR method and sequence analysis. Results: The CSF sample collected was taken into distilled water considering the possibility of transformation of trophozoites to intermediate form and incubated at 37 °C for 1 to 2 h, and pear-shaped non-permanent flagellated forms were observed in the direct microscopic examination, and molecular typing was performed to confirm the diagnosis. This study was a comprehensive case of N. fowleri whose etiological agent was isolated and confirmed by real-time PCR in Turkey. Conclusion: Clinician awareness would be the key factor in correctly diagnosing PAM. It is also recommended to investigate all likely environmental water sources in Turkey for more detailed information on the distribution and molecular identification of Naegleria species, ultimately to evaluate the potential pathogenic threat to human health and to develop strategies to combat such threats. [ABSTRACT FROM AUTHOR]

Free-living amoebas (FLAs) can cause neurological and ocular complications in humans. Water supplies play a critical role in transmitting FLAs to humans. The aim of the present study was to investigate the presence of FLAs in various aquatic sources including drinking water, stagnant water, and surface water in Alborz province, northern Iran, using morphological and molecular techniques. A total of 70 water samples were collected from 34 drinking waters, 23 surface waters, and 13 stagnant waters. Filtration and cultivation were employed to isolate FLAs. PCR assay was applied by using the genus-specific primers on positive samples. Pathogenicity tests (osmo-and thermo-tolerance properties) were performed for Acanthamoeba spp., positive sample. Considering the morphological criteria, four positive samples of Acanthamoeba sp., three Vermamoeba sp., two mixed Vermamoeba sp. with Vahlkamfiids, and one mixed Acanthamoeba sp. with Vahlkamfiids were isolated. Five Acanthamoeba sp. isolates were amplified using the JDP primer pairs. Among them, two genotypes, T4 (three isolates) and T5 (two isolates) corresponding to A. lenticulata, were identified. Four V. vermiformis samples were confirmed using the sequencing. This study highlighted the occurrence of potentially pathogenic waterborne FLAs in water habitats associated with high human activity. The results of such research on the prevalence of FLAs, as a human hazard, should be communicated to health policymakers. [ABSTRACT FROM AUTHOR]

Naegleria fowleri is a pathogenic, free‐living amoeba that causes primary amebic meningoencephalitis (PAM), a highly fatal disease of the central nervous system. N. fowleri demonstrates three forms: the trophozoite, flagellate, and cyst. Most studies have focused on the trophozoite limiting information on the cyst. The present study examined the ability of cysts to attach to, excyst into the trophozoite form, and destroy cell cultures. Additionally, the study assessed the ability of cysts to cause PAM in a murine model. The results demonstrated that exposure to cysts and transformation into trophozoites resulted in destruction of cell cultures. Specifically, the mixed glial cells exhibited an increase in lactate dehydrogenase (LDH) release compared with cells without cyst exposure. On day eight postexposure, there was a nearly fourfold increase in LDH. The cysts of N. fowleri were shown not to be infective in vivo in a murine model. The mediation of the encystment process by the intracellular concentration of cAMP was also investigated. Trophozoites were treated with dipyridamole, an inhibitor of cAMP‐specific phosphodiesterases. Dipyridamole increased the rate of encystment by nearly twofold and increased the intracellular concentration of cAMP in cysts by nearly sixfold throughout this period suggesting that cAMP is a mediator of encystment for N. fowleri. [ABSTRACT FROM AUTHOR]

Purpose: Acanthamoeba spp. are free-living amoebas with worldwide distribution and play an important role as disease-causing agents in humans. Drug inability to completely eradicate these parasites along with their toxic effects suggest urgent need for new antimicrobials. Nisin is a natural antimicrobial peptide produced by Lactococcus lactis. Nisin is also the only bacteriocin approved for use in food preservation. In this work, we analyzed the effect of nisin on the growth of Acanthamoeba castellanii trophozoites. Methods: A total of 8 × 104 trophozoites were exposed to increasing concentrations of nisin to determine its activity. Changes in cell membrane and cellular cycle of trophozoites were investigated by flow cytometry, and nisin cytotoxicity in mammalian cells was evaluated in L929 cells by MTT method. Results: After 24 h exposure to increasing nisin concentrations, an IC50 of 4493.2 IU mL−1 was obtained for A. castellanii trophozoites. However, after 72 h a recovery in amoebic growth was observed, and it was no longer possible to determine IC50. Flow cytometry analysis showed that nisin has no effect on the membrane integrity. Treatment with nisin induced cell-cycle arrest during G1 and S phases in A. castellanii trophozoites, which recovered their growth after 72 h. Conclusion: This is one of the first studies showing the effect of internationally approved nisin against A. castellanii trophozoites. Nisin caused cell-cycle arrest in trophozoites, momentarily interfering with the DNA replication process. The data highlight the amoebostatic activity of nisin, and suggest its use as an adjuvant for the treatment of infections caused by Acanthamoeba spp. [ABSTRACT FROM AUTHOR]

Objective: Acanthamoeba is a free-living and opportunistic amoeba that the potential of this parasite to convert to a cyst, making its treatment difficult. In this study, we investigated the effect of microwave radiation on Acanthamoeba cysts in vitro. Methods: Acanthamoeba cysts were reproduced in the culture medium. We divided 16 test tubes containing cysts into two groups. The contents of 8 tubes were irradiated with continuous microwave in the time range of 0-120 s, and the next 8 tubes were irradiated with repetitive microwave in the range of 0-360 s. The mortality of cysts at the end of irradiation was recorded and compared with control. Results: Continuous and repetitive irradiation of microwave resulted in mortality of 100 (%) of Acanthamoeba cysts during 120 and 360 seconds, respectively. In continuous mode, parasite mortality in all groups was significantly different from the control group (P

Background & objectives: Impaired immune system provides favorable conditions for colonization by Acanthamoeba in the human body. In this case control study, we compared the molecular and culture methods in identifying Acanthamoeba in the nasal and oral secretions of HIV+/HIV­ human. Methods: In a current case control study, nasal and oral discharge of 53, HIV+ patients and 53, HIV­ people were evaluated. The nasal and oral secretions of each patient were prepared by sterile swabs and transferred to the laboratory. All samples were cultured but only the positive samples used for molecular analysis. Results: By cultivation method, of the 53, HIV+ patients, a total of 11 samples, including 5 nasal and 6 oral samples, were contaminated with Acanthamoeba. Of the 53, HIV­­ people, 3 samples of nasal discharge were contaminated with this parasite. The molecular method approved the contamination of 10 samples, including 5 oral and 5 nasal samples from HIV+ patients with this parasite. Statistical analysis showed the rate of infection in HIV+ patients was significantly different compared to HIV­ people Conclusion: The results of the current study showed that the rate of Acanthamoeba infection in HIV+ patients was higher than that of HIV- individuals. Also, considering that in the control group (HIV- individuals) only the nasal discharge were infected with the parasite, it seems that in the case group (HIV + patients) the infection of the oral discharge with the parasites is due to the entry of its cysts into the nose and transmission to the mouth

Introduction: Free-living amoeba is ubiquitous in fresh water, mud, and moist soil; although seldom pathogenic to humans, Naegleria fowleri , Acanthamoeba spp., and Balamuthia spp. are known to cause infections of the central nervous system., Methods: We report two interesting cases, both of which presented with a rapid-onset and fulminant course. The first case details a 36-year-old male with a history of surgically corrected nasal bone fracture 15 years back, who presented with acute onset of fever, headache and convulsions., Results: Direct smears of cerebrospinal fluid (CSF) revealed motile trophozoites resembling N. fowleri and were later confirmed by molecular diagnosis (polymerase chain reaction [PCR]). Subsequently, the source was identified as water used for religious purposes. Unfortunately, the patient succumbed to cardiac arrest. The second case is that of a 4-year-old boy from Palakkad district, Kerala, who presented with sudden onset of nasal discharge, fever, headache, vomiting, seizures and altered sensorium. His CSF smear examination showed motile trophozoites resembling Acanthamoeba spp. While undergoing treatment, he seized thrice before going into cardiac arrest. Despite extensive resuscitation measures, the child could not be revived and passed away. The postmortem CSF sample sent for molecular analysis confirmed infection by Acanthamoeba spp., Conclusion: Rapid progression and lack of definite treatment options make this a highly fatal condition. Although clinical presentations of both patients were indicative of pyogenic meningitis, parasitic infection was suspected when the CSF was found turbid with no bacteria, high protein, and low sugar. High index of suspicion helped us to get an early preliminary diagnosis from direct microscopy., Competing Interests: There are no conflicts of interest., (Copyright: © 2024 Tropical Parasitology.)

Background: Pathogenic free-living amoeba (FLA) such as Acanthamoeba spp., Naegleria fowleri, and Balamuthia mandrillaris are causative agents of fatal amoebic encephalitis/meningoencephalitis. The diagnosis of such infections is challenging due to a lack of clinical suspicion and expertise in microscopic identification. We evaluated the performance of molecular assays for the timely and accurate detection of FLA-causing central nervous system (CNS) afflictions. Methods: This study included samples from 156 patients with suspected encephalitis/meningoencephalitis, including 149 cerebrospinal fluid (CSF) samples, 5 brain tissue biopsies, and 2 brain abscess samples. All the samples were subjected to PCR-based detection of Acanthamoeba spp., N. fowleri, and B. mandrillaris. The diagnostic characteristics and the inter-rater reliability scores were evaluated for parasite-specific polymerase chain reaction (PCR) using culture on non-nutrient agar (NNA)/microscopy or histopathological examination as a confirmatory test for Acanthamoeba spp. and N. fowleri and histopathology for B. mandrillaris. Results: We detected 11 samples positive for FLA, including 6 Acanthamoeba spp., 3 B. mandrillaris, and 2 N. fowleri. Furthermore, all 11 samples were positive according to the confirmatory tests, i.e., culture on NNA/microscopy/histopathology in the case of Acanthamoeba spp. and N. fowleri and histopathology of tissue biopsies for B. mandrillaris. The inter-rater reliability between the PCRs and the confirmatory tests for the detection of Acanthamoeba spp., N. fowleri, and B. mandrillaris was 100%. Conclusions: The PCR-based detection of FLA in patients suspected of encephalitis/meningoencephalitis was found to be fast, efficient, and reliable in our study. We suggest the use of these PCRs in laboratories to obtain additional data on their efficiency in diagnosing FLA infections of the CNS. The present study was conducted with a small sample size of 156 patient samples, and we found only six Acanthamoeba spp., three B. mandrillaris, and two N. fowleri. The present study should be conducted on a larger sample size for better evaluation of the primer pairs.

Acanthamoeba is a free-living protozoan known to cause keratitis most commonly, especially among contact lens wearers. Treatment of Acanthamoeba keratitis is challenging as Acanthamoeba can encyst from the active form, a trophozoite, into a hibernating cyst that is refractory to antibiotics and difficult to kill; therefore, there is a need for more effective anti-amoebic strategies. In this study, we have evaluated the anti-amoebic activity of the antimicrobial peptide mimic RK-758 against Acanthamoeba castellanii. RK-758 peptidomimetic was subjected to biological assays to investigate its amoebicidal, amoebistatic, anti-encystation, and anti-excystation effects on A. castellanii. The anti-amoebic activity of the peptide mimic RK-758 was compared with chlorhexidine against the Acanthamoeba castellanii ATCC30868 and Acanthamoeba castellanii 044 (a clinical strain) with the concentrations of both ranging from 125 µM down to 7.81 µM. All experiments were performed in duplicate with three independent replicates. The data were represented as mean ± SE and analysed using a two-sample t-test and two-tailed distributions. A p < 0.05 was considered statistically significant. The peptidomimetic RK-758 had anti-Acanthamoeba activity against both trophozoites and cysts in a dose-dependent manner. The RK-758 had amoebicidal and growth inhibitory activities of ≥50% at a concentration between 125 µM and 15.6 µM against the trophozoites of both Acanthamoeba strains. Inhibitory effects on the cyst formation and trophozoite re-emergence from cysts were noted at similar concentrations. Chlorhexidine had 50% activity at 7.81 µM and above against the trophozoites and cysts of both strains. In the haemolysis assay, the RK-758 lysed horse RBCs at concentrations greater than 50 µM whereas lysis occurred at concentrations greater than 125 µM for the chlorhexidine. The peptidomimetic RK-758, therefore, has activity against both the trophozoite and cyst forms of Acanthamoeba and has the potential to be further developed as an anti-microbial agent against Acanthamoeba. RK-758 may also have use as an anti-amoebic disinfectant in contact lens solutions.

Arginine-rich peptides can have broad-spectrum anti-bacterial and anti-fungal activities. Polyhomoarginine consists of highly cationic residues which can act on the negatively charged microbial cell membranes. Acanthamoeba is a free-living protozoan known to cause a rare corneal infection which is difficult to diagnose and treat. This study evaluated the activity of the polyhomoarginines against Acanthamoeba castellanii. Acanthamoeba amoebicidal, amoebistatic, encystation and excystment assays were performed using protocols described in the literature. The activity of polyhomoarginines (PHAs) of different lengths (10 to 400 residues) was measured against the trophozoites and cysts of Acanthamoeba castellanii ATCC30868 in concentrations ranging from 0.93 μM to 15 μM. Data were represented as mean ± SE and analysed using one-way ANOVA. Overall, PHAs demonstrated good anti-acanthamoeba activity against both trophozoites and cysts. PHA 30 reduced the number of viable trophozoites by 99%, inhibited the formation of cysts by 96% and the emergence of trophozoites from cysts by 67% at 3.75 μM. PHA 10 was similarly active, but at a slightly higher concentration of 15 μM, reducing the numbers of viable trophozoites by 98%, inhibiting cyst formation by 84% and preventing the emergence of trophozoites from cysts by 99%. At their greatest anti-amoeba concentrations, PHA 10 gave only 8% haemolysis at 15 μM while PHA 30 gave Acanthamoeba castellanii and was non-toxic at its most active concentrations. This implies that polyhomoarginines can be developed into a potential therapeutic agent for Acanthamoeba keratitis. However, there is a need to carry out further pre-clinical and then in vivo experiments in the AK animal model.

Background and purpose: Acanthamoeba spp. are an opportunistic protozoan in environmental sources that can cause respiratory infection and keratitis. The present study was conducted to determine the prevalence and genotypes of Acanthamoeba in rural drinking water sources in Kashan and Aran-Bidgol, Iran. Materials and methods: In this study, 162 samples from 54 drinking water sources were collected in 2017. After samples filtration, they were cultured on non-nutrient agar to isolate free-living amoeba. After DNA extraction, the Polymerase Chain Reaction using JDP1, JDP2 primers was performed to detect Acanthamoeba spp. and finally the genotype of eight isolates was determined. Data analysis was done in SPSS applying Chi-square and Fisher’s exact test. Results: Among the samples 35.2% were found to be contaminated with free-living amoeba, among which 19 isolates (11.7%) were confirmed as Acanthamoeba. The rates of free-living amoeba and Acanthamoba were 35.4% and 11.8%, respectively, in Kashan, which were higher than those in Aran-Bidgol. Frequency of free-living amoeba in Qanat, well, and spring was 55.6%, 30.2%, and 26.7%, respectively. Acanthamoeba contamination rates were 13.9%, 11.5% and 10%, respectively. Acanthamoeba contamination rate was higher in surface wells than that in deep wells. The genotype of all Acanthamoeba isolates belonged to T4. Conclusion: This study revealed that drinking water sources were contaminated with free-living amoeba and Acanthamoeba. All isolates were T4 genotype, therefore, these sources could be considered as a risk factor for public health.

Abstract Recently, indoor swimming pool activities have increased to promote health‐enhancing physical activities, which require establishing suitable protocols for disinfection and water quality control. Normally, the assessment of the microbial quality of the water in the pools only considers the presence of different bacteria. However, other less frequent but more resistant pathogens, such as free‐living amoebas (FLA), are not contemplated in both existing recommendation and research activities. FLA represent a relevant human health risk, not only due to their pathogenicity but also due to the ability to act as vehicles of other pathogens, such as bacteria. Therefore, this work aimed to study the physicochemical characteristics and the occurrence of potentially pathogenic FLA and bacteria in water samples from 20 public indoor swimming facilities in Northern Portugal. Our results showed that some swimming pools presented levels of pH, free chlorine, and conductivity out of the recommended limits. Pathogenic FLA species were detected in two of the facilities under study, where we also report the presence of both, FLA and pathogenic bacteria. Our findings evidence the need to assess the occurrence of FLA and their existence in the same environmental niche as pathogenic bacteria in swimming pool facilities worldwide and to establish recommendations to safeguard the health of the users.

In industrial water systems, the occurrence of biofilm-associated pathogenic free-living amoebae (FLA) such as Naegleria fowleri is a potential hygienic problem, and factors associated with its occurrence remain poorly understood. This study aimed to evaluate the impact of four cooling circuit materials on the growth of N. fowleri in a freshwater biofilm formed at 42°C and under a hydrodynamic shear rate of 17 s -1 (laminar flow): polyvinyl chloride, stainless steel, brass, and titanium. Colonization of the freshwater biofilms by N. fowleri was found to be effective on polyvinyl chloride, stainless steel, and titanium. For these three materials, the ratio of (bacterial prey)/(amoeba) was found to control the growth of N. fowleri . All materials taken together, a maximum specific growth rate of 0.18 ± 0.07 h -1 was associated with a generation time of ~4 h. In contrast, no significant colonization of N. fowleri was found on brass. Therefore, the contribution of copper is strongly suspected., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Goudot, Mathieu, Herbelin, Soreau and Jorand.)