Pathogenic Free-living Amoebae Research Articles

Amoebicidal effect of chlorine dioxide gas against pathogenic Naegleria fowleri and Acanthamoeba polyphaga.

The pathogenic free-living amoebae, Naegleria fowleri and Acanthamoeba polyphaga, are found in freshwater, soil, and unchlorinated or minimally chlorinated swimming pools. N. fowleri and A. polyphaga are becoming problematic as water leisure activities and drinking water are sources of infection. Chlorine dioxide (ClO2) gas is a potent disinfectant that is relatively harmless to humans at the concentration used for disinfection. In this study, we examined the amoebicidal effects of ClO2 gas on N. fowleri and A. polyphaga. These amoebae were exposed to ClO2 gas from a ready-to-use product (0.36 ppmv/h) for 12, 24, 36, and 48h. Microscopic examination showed that the viability of N. fowleri and A. polyphaga was effectively inhibited by treatment with ClO2 gas in a time-dependent manner. The growth of N. fowleri and A. polyphaga exposed to ClO2 gas for 36h was completely inhibited. In both cases, the mRNA levels of their respective actin genes were significantly reduced following treatment with ClO2 gas. ClO2 gas has an amoebicidal effect on N. fowleri and A. polyphaga. Therefore, ClO2 gas has been proposed as an effective agent for the prevention and control of pathogenic free-living amoeba contamination.

Growth dynamic of biofilm-associated Naegleria fowleri in freshwater on various materials.

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.

Global prevalence of potentially pathogenic free-living amoebae in sewage and sewage-related environments-systematic review with meta-analysis.

Free-living amoebae (FLA) include amphizoic microorganisms important in public health, widely isolated from air, water, and soil. However, its occurrence in sewage-related environments still needs to be systematically documented. This study summarizes the occurrence of FLA in sewage-related environments through a systematic review with meta-analysis. A total of 1983 scientific article were retrieved from different databases, of which 35 were selected and analyzed using a random effects forest plot model with a 95% confidence interval (IC). The pooled overall prevalence of FLA in sewage across 12 countries was 68.96% (95% IC = 58.5-79.42). Subgroup analysis indicates high prevalence in all environments analyzed, including sewage water from the sewage treatment plant (81.19%), treated sewage water (75.57%), sewage-contaminated water (67.70%), sediment contaminated by sewage (48.91%), and sewage water (47.84%). Prevalence values of Acanthamoeba spp., Hartmanella/Vermamoeba spp., and Naegleria spp. are 47.48%, 28.24%, and 16.69%, respectively. Analyzing the species level, the distribution is as follows: Acanthamoeba palestinensis (88%), A. castellanii (23.74%), A. astronyxis (19.18%), A. polyphaga (13.59%), A. culbertsoni (12.5%), A. stevensoni (8.33%), A. tubiashi (4.35%) and A. hatchetti (1.1%), Naegleria fowleri (28.4%), N. gruberi (25%), N. clarki (8.33%), N. australiensis (4.89%) and N. italica (4.29%), Hartmannella/Vermamoeba exundans (40%) and H.V. vermiform (32.61%). Overall, our findings indicate a high risk associated with sewage-related environments, as the prevalence of FLA, including pathogenic strains, is high, even in treated sewage water. The findings of this study may be valuable both for risk remediation actions against amoebic infections and for future research endeavors.

Influence of salt and temperature in the growth of pathogenic free-living amoebae.

Free-living amoebae are an extensive group of protistans that can be found in a wide variety of environments. Among them, the Acanthamoeba genus and Naegleria fowleri stand out as two of the most pathogenic amoebae and with a higher number of reported cases. N. fowleri is mainly found in warm freshwater water bodies whereas amoebae of the Acanthamoeba genus are broadly distributed through natural and anthropogenic environments. In this regard, the management and the control of the amoebic populations in swimming pools has become a major public health challenge for institutions. The aim of this work was to evaluate the growth pattern of trophozoites of A. griffini and N. fowleri at different temperatures and salt concentrations. Our results showed that A. griffini resisted a higher concentration of salt than N. fowleri. Moreover, no trophozoites could withstand the salt levels of the sea in in vitro conditions. This work supports the contention that salinity could represent an important and useful tool for the control of the most pathogenic amoebic populations in recreational water bodies.

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

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

Metabarcoding of protozoa and helminth in black-necked cranes: a high prevalence of parasites and free-living amoebae.

Parasites and free-living amoebae (FLA) are common pathogens that pose threats to wildlife and humans. The black-necked crane (Grus nigricollis) is a near-threatened species and there is a shortage of research on its parasite diversity. Our study aimed to use noninvasive methods to detect intestinal parasites and pathogenic FLA in G. nigricollis using high-throughput sequencing (HTS) based on the 18S rDNA V9 region. A total of 38 fresh fecal samples were collected in Dashanbao, China, during the overwintering period (early-, middle I-, middle II-, and late-winter). Based on the 18S data, eight genera of parasites were identified, including three protozoan parasites: Eimeria sp. (92.1%) was the dominant parasite, followed by Tetratrichomonas sp. (36.8%) and Theileria sp. (2.6%). Five genera of helminths were found: Echinostoma sp. (100%), Posthodiplostomum sp. (50.0%), Euryhelmis sp. (26.3%), Eucoleus sp. (50.0%), and Halomonhystera sp. (2.6%). Additionally, eight genera of FLA were detected, including the known pathogens Acanthamoeba spp. (n=13) and Allovahlkampfia spp. (n=3). Specific PCRs were used to further identify the species of some parasites and FLA. Furthermore, the 18S data indicated significant changes in the relative abundance and genus diversity of the protozoan parasites and FLA among the four periods. These results underscore the importance of long-term monitoring of pathogens in black-necked cranes to protect this near-endangered species.

Antiamoebic activities of flavonoids against pathogenic free-living amoebae, Naegleria fowleri and Acanthamoeba species.

Free-living amoebae (FLA) rarely cause human infections but can invoke fatal infections in the central nervous system (CNS). No consensus treatment has been established for FLA infections of the CNS, emphasizing the urgent need to discover or develop safe and effective drugs. Flavonoids, natural compounds from plants and plant-derived products, are known to have antiprotozoan activities against several pathogenic protozoa parasites. The anti-FLA activity of flavonoids has also been proposed, while their antiamoebic activity for FLA needs to be emperically determined. We herein evaluated the antiamoebic activities of 18 flavonoids against Naegleria fowleri and Acanthamoeba species which included A. castellanii and A. polyphaga. These flavonoids showed different profiles of antiamoebic activity against N. fowleri and Acanthamoeba species. Demethoxycurcumin, kaempferol, resveratrol, and silybin (A+B) showed in vitro antiamoebic activity against both N. fowleri and Acanthamoeba species. Apigenin, costunolide, (‒)-epicatechin, (‒)-epigallocatechin, rosmarinic acid, and (‒)-trans-caryophyllene showed selective antiamoebic activity for Acanthamoeba species. Luteolin was more effective for N. fowleri. However, afzelin, berberine, (±)-catechin, chelerythrine, genistein, (+)-pinostrobin, and quercetin did not exhibit antiamoebic activity against the amoeba species. They neither showed selective antiamoebic activity with significant cytotoxicity to C6 glial cells. Our results provide a basis for the anti-FLA activity of flavonoids, which can be applied to develope alternative or supplemental therapeutic agents for FLA infections of the CNS.

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

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

Glucose metabolism in the pathogenic free-living amoebae: Tempting targets for treatment development.

Pathogenic free-living amoebae (pFLA) are single-celled eukaryotes responsible for causing intractable infections with high morbidity and mortality in humans and animals. Current therapeutic approaches include cocktails of antibiotic, antifungal, and antimicrobial compounds. Unfortunately, the efficacy of these can be limited, driving the need for the discovery of new treatments. Pan anti-amebic agents would be ideal; however, identifying these agents has been a challenge, likely due to the limited evolutionary relatedness of the different pFLA. Here, we discuss the potential of targeting amoebae glucose metabolic pathways as the differences between pFLA and humans suggest specific inhibitors could be developed as leads for new therapeutics.

Shotgun Kinetic Target-Guided Synthesis Approach Enables the Discovery of Small-Molecule Inhibitors against Pathogenic Free-Living Amoeba Glucokinases.

Pathogenic free-living amoebae (pFLA) can cause life-threatening central nervous system (CNS) infections and warrant the investigation of new chemical agents to combat the rise of infection from these pathogens. Naegleria fowleri glucokinase (NfGlck), a key metabolic enzyme involved in generating glucose-6-phosphate, was previously identified as a potential target due to its limited sequence similarity with human Glck (HsGlck). Herein, we used our previously demonstrated multifragment kinetic target-guided synthesis (KTGS) screening strategy to identify inhibitors against pFLA glucokinases. Unlike the majority of previous KTGS reports, our current study implements a "shotgun" approach, where fragments were not biased by predetermined binding potentials. The study resulted in the identification of 12 inhibitors against 3 pFLA glucokinase enzymes─NfGlck, Balamuthia mandrillaris Glck (BmGlck), and Acanthamoeba castellanii Glck (AcGlck). This work demonstrates the utility of KTGS to identify small-molecule binders for biological targets where resolved X-ray crystal structures are not readily accessible.

Characterization of the extracellular vesicles, ultrastructural morphology, and intercellular interactions of multiple clinical isolates of the brain-eating amoeba, Naegleria fowleri.

As global temperatures rise to unprecedented historic levels, so too do the latitudes of habitable niches for the pathogenic free-living amoeba, Naegleria fowleri. This opportunistic parasite causes a rare, but >97% fatal, neurological infection called primary amoebic meningoencephalitis. Despite its lethality, this parasite remains one of the most neglected and understudied parasitic protozoans. To better understand amoeboid intercellular communication, we elucidate the structure, proteome, and potential secretion mechanisms of amoeba-derived extracellular vesicles (EVs), which are membrane-bound communication apparatuses that relay messages and can be used as biomarkers for diagnostics in various diseases. Herein we propose that N. fowleri secretes EVs in clusters from the plasma membrane, from multivesicular bodies, and via beading of thin filaments extruding from the membrane. Uptake assays demonstrate that EVs are taken up by other amoebae and mammalian cells, and we observed a real-time increase in metabolic activity for mammalian cells exposed to EVs from amoebae. Proteomic analysis revealed >2,000 proteins within the N. fowleri-secreted EVs, providing targets for the development of diagnostics or therapeutics. Our work expands the knowledge of intercellular interactions among these amoebae and subsequently deepens the understanding of the mechanistic basis of PAM.

Repurposing of Nitroxoline as an Alternative Primary Amoebic Meningoencephalitis Treatment.

Among the pathogenic free-living amoebae (FLA), Naegleria fowleri is the etiological agent of a fatal disease known as primary amoebic meningoencephalitis (PAM). Once infection begins, the lesions generated in the central nervous system (CNS) result in the onset of symptoms leading to death in a short period of time. Currently, there is no standardized treatment against the infection, which, due to the high virulence of the parasite, results in a high case fatality rate (>97%). Therefore, it is essential to search for new therapeutic sources that can generate a rapid elimination of the parasite. In recent years, there have already been several successful examples of drug repurposing, such as Nitroxoline, for which, in addition to its known bioactive properties, anti-Balamuthia activity has recently been described. Following this approach, the anti-Naegleria activity of Nitroxoline was tested. Nitroxoline displayed low micromolar activity against two different strains of N. fowleri trophozoites (IC50 values of 1.63 ± 0.37 µM and 1.17 ± 0.21 µM) and against cyst stages (IC50 of 1.26 ± 0.42 μM). The potent anti-parasitic activity compared to the toxicity produced (selectivity index of 3.78 and 5.25, respectively) in murine macrophages and human cell lines (reported in previous studies), together with the induction of programmed cell death (PCD)-related events in N. fowleri make Nitroxoline a great candidate for an alternative PAM treatment.

Molecular identification of Acanthamoeba spp., Balamuthia mandrillaris and Naegleria fowleri in soil samples using quantitative real-time PCR assay in Turkey; Hidden danger in the soil!

Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri are pathogenic free-living amoeba (FLA) and are commonly found in the environment, particularly soil. This pathogenic FLA causes central nervous system-affecting granulomatous amebic encephalitis (GAE) or primary amebic meningoencephalitis (PAM) and can also cause keratitis and skin infections. In the present study, we aimed to determine the quantitative concentration of Acanthamoeba spp., B. mandrillaris, and N. fowleri in soil samples collected from places where human contact is high by using a qPCR assay in Izmir, Turkey. A total of 45.71% (n = 16) of Acanthamoeba spp., 20% (n = 7) of B. mandrillaris, and 17.4% (n = 6) of N. fowleri were detected in five different soil sources by the qPCR assay. The quantitative concentration of Acanthamoeba spp., B. mandrillaris, and N. fowleri in various soil sources was calculated at 10 × 105 - 6 × 102, 47 × 104 to 39 × 103, and 9 × 103 – 8 × 102 plasmid copies/gr, respectively. While the highest quantitative concentration of Acanthamoeba spp. and B. mandrillaris was determined in garden soil samples, N. fowleri was detected in potting soil samples. Three different genotypes T2 (18.75%), T4 (56.25%), and T5 (25%) were identified from Acanthamoeba-positive soil samples. Acanthamoeba T4 genotype was the most frequently detected genotype from soil samples and is also the most common genotype to cause infection in humans and animals. To the best of our knowledge, the present study is the first study to identify genotype T5 in soil samples from Turkey. In conclusion, people and especially children should be aware of the hidden danger in the garden and potting soil samples that come into contact most frequently. Public health awareness should be raised about human infections that may be encountered due to contact with the soil. Public health specialists should raise awareness about this hidden danger in soil.

Identification of novel anti-amoebic pharmacophores from kinase inhibitor chemotypes.

Acanthamoeba species, Naegleria fowleri, and Balamuthia mandrillaris are opportunistic pathogens that cause a range of brain, skin, eye, and disseminated diseases in humans and animals. These pathogenic free-living amoebae (pFLA) are commonly misdiagnosed and have sub-optimal treatment regimens which contribute to the extremely high mortality rates (>90%) when they infect the central nervous system. To address the unmet medical need for effective therapeutics, we screened kinase inhibitor chemotypes against three pFLA using phenotypic drug assays involving CellTiter-Glo 2.0. Herein, we report the activity of the compounds against the trophozoite stage of each of the three amoebae, ranging from nanomolar to low micromolar potency. The most potent compounds that were identified from this screening effort were: 2d (A. castellanii EC50: 0.92 ± 0.3 μM; and N. fowleri EC50: 0.43 ± 0.13 μM), 1c and 2b (N. fowleri EC50s: <0.63 μM, and 0.3 ± 0.21 μM), and 4b and 7b (B. mandrillaris EC50s: 1.0 ± 0.12 μM, and 1.4 ± 0.17 μM, respectively). With several of these pharmacophores already possessing blood-brain barrier (BBB) permeability properties, or are predicted to penetrate the BBB, these hits present novel starting points for optimization as future treatments for pFLA-caused diseases.

Naegleria australiensis isolated from a wastewater treatment station in Santiago Island, Cape Verde.

Despite the Naegleria genus being isolated from different natural environments such as water, soil, and air, not all Naegleria species are capable of causing infections in humans, and they are capable of completing their life cycle in environmental niches. However, the presence of this genus may suggest the existence of one of the highly pathogenic free-living amoeba (FLA) species: Naegleria fowleri or the brain-eating amoeba. This facultative parasitic protozoon represents a risk to public health, mainly related to domestic and agricultural waters. In this research, our main objective was to determine the existence of pathogenic protozoa in the Santa Cruz wastewater treatment plant, Santiago Island. Using 5 L of water we confirmed the presence of potentially pathogenic Naegleria australiensis, being the first report on Naegleria species in Cape Verde. This fact demonstrates the low efficiency in the treatment of wastewater and, consequently, a potential threat to public health. Nevertheless, more studies will be needed for the prevention and control of possible infections in this Macaronesian country.

De Novo Transcriptome Profiling of Naegleria fowleri Trophozoites and Cysts via RNA Sequencing

Naegleria fowleri is a pathogenic free-living amoeba, commonly found around the world in warm, fresh water and soil. N. fowleri trophozoites can infect humans by entering the brain through the nose and causing usually fatal primary amebic meningoencephalitis (PAM). Trophozoites can encyst to survive under unfavorable conditions such as cold temperature, starvation, and desiccation. Recent technological advances in genomics and bioinformatics have provided unique opportunities for the identification and pre-validation of pathogen-related and environmental resistance through improved understanding of the biology of pathogenic N. fowleri trophozoites and cysts at a molecular level. However, genomic and transcriptomic data on differential expression genes (DEGs) between trophozoites and cysts of N. fowleri are very limited. Here, we report transcriptome Illumina RNA sequencing (RNA-seq) for N. fowleri trophozoites and cysts and de novo transcriptome assembly. RNA-seq libraries were generated from RNA extracted from N. fowleri sampled from cysts, and a reference transcriptome was generated through the assembly of trophozoite data. In the database, the assembly procedure resulted in 42,220 contigs with a mean length of 11,254 nucleotides and a C+G content of 37.21%. RNA sequencing showed that 146 genes in cysts of N. fowleri indicated 2-fold upregulation in comparison with trophozoites of N. fowleri, and 163 genes were downregulated; these genes were found to participate in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. The KEGG pathway included metabolic (131 sequences) and genetic information processing (66 sequences), cellular processing (43 sequences), environmental information processing (22 sequences), and organismal system (20 sequences) pathways. On the other hand, an analysis of 11,254 sequences via the Gene Ontology database showed that their annotations contained 1069 biological processes including the cellular process (228 sequences) and metabolic process (214 sequences); 923 cellular components including cells (240 sequences) and cell parts (225 sequences); and 415 molecular functions including catalytic activities (195 sequences) and binding processes (186 sequences). Differential expression levels increased in cysts of N. fowleri compared to trophozoites of N. fowleri, which were mainly categorized as serine/threonine protease, kinase, and lipid metabolism-related proteins. These results may provide new insights into pathogen-related genes or environment-resistant genes in the pathogenesis of N. fowleri.

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

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

Molecular Diagnosis of Encephalitis/Meningoencephalitis Caused by Free-Living Amoebae from a Tertiary Center in India.

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. 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. 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%. 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.

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

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

Exploring LSU and ITS rDNA Sequences for Acanthamoeba Identification and Phylogeny.

The identification and classification of strains of Acanthamoeba, a potentially pathogenic ubiquitous free-living amoeba, are largely based on the analysis of 18S rDNA sequences, currently delineating 23 genotypes, T1 to T23. In this study, the sequences of the ITS region, i.e., the 5.8S rDNA and the two internal transcribed spacers (ITS-1 and ITS-2), and those of the large subunit (LSU) rDNA of Acanthamoeba were recovered from amoeba genomes; the sequences are available in GenBank. The complete ITS–LSU sequences could be obtained for 15 strains belonging to 7 distinct lineages (T4A, T4D, T4F, T4G, T2, T5, and T18), and the site of the hidden break producing the 26Sα and 26Sβ was identified. For the other lines, either the LSU is partial (T2/T6, T7) or the ITS is fragmentary (T7, T10, T22). It is noteworthy that a number of sequences assigned to fungi turned out to actually be Acanthamoeba, only some of which could be affiliated with known genotypes. Analysis of the obtained sequences indicates that both ITS and LSU are promising for diagnostic and phylogenetic purposes.