Warm Freshwater Research Articles

Exploring ice melting dynamics in beverageware

The ice-melting process inside water-filled beverageware is studied experimentally, theoretically, and numerically. An ice body is immersed in warm fresh water in different-shaped glass cups. The ice body eventually melts due to heat transfer from the warmer fluid to the ice. As the ice melts and cools the surrounding fluid, it promotes complex time-dependent laminar flows characterized by a vertical downward jet beneath the ice body and rotating convective cells in the fluid domain. Experimental results obtained through dye visualization, particle image velocity, and local temperature measurements show that the size and number of the rotating cells, jet velocity, and melting time of the ice depend significantly on the shape of the beverageware. A one-dimensional analytical solution reproduces qualitatively the hydrodynamical and thermal boundary layers close to the ice body. Further, a two-dimensional numerical model correctly captures the main features of the experimental observations.

Naegleria fowleri 98 Percent Fatal a Comprehensive Survey

Naegleria fowleri, colloquially termed the "brain-eating amoeba," presents a formidable public health concern due to its astonishingly high fatality rate of 98%. This abstract provides a thorough examination of the various aspects surrounding Naegleria fowleri, encompassing its morphology, life cycle, transmission routes, clinical manifestations, diagnostic methods, treatment strategies, and preventive measures. Naegleria fowleri, an amoeboid protist, thrives in warm freshwater environments, predominantly in regions with elevated temperatures. Infections occur when contaminated water enters the nasal passages, leading to primary amebic meningoencephalitis (PAM), an acute and often fatal condition affecting the central nervous system. The clinical course of PAM is rapid, with symptoms escalating from initial flu-like manifestations to severe neurological impairment within days. Diagnosis of PAM remains challenging, relying on specialized laboratory techniques such as polymerase chain reaction (PCR) and immunofluorescence assays. Despite efforts to develop effective treatments, mortality rates remain alarmingly high, emphasizing the critical importance of preventive measures. Avoiding freshwater activities in warm, stagnant water bodies, utilizing nose clips or plugs during water-related activities, and ensuring proper disinfection of recreational water sources are key strategies in mitigating the risk of the risk of Naegleria fowleri infections and reducing its devastating impact on public health.

Redefining piscine lactococcosis.

Lactococcosis affects a broad range of host species, including fish from cold, temperate, and warm freshwater or marine environments, as well as several terrestrial animals, including humans. As such, lactococcosis is a disease of concern for animal and ecosystem health. The disease is endemic in European and Asian aquaculture but is rapidly encroaching on ecologically and economically important fish populations across the Americas. Piscine lactococcosis is difficult to manage, with issues of vaccine escape, ineffective antimicrobial treatment, and the development of carrier fish or biofilms leading to recurrent outbreaks. Our understanding of the disease is also widely outdated. The accepted etiologic agent of lactococcosis is Lactococcus garvieae. However, historical misidentification has masked contributions from two additional species, L. petauri and L. formosensis, which are indistinguishable from L. garvieae by common diagnostic methods. This work is the first comprehensive characterization of all three agents and provides direct recommendations for species-specific diagnosis and management.

In silico discovery of diagnostic/vaccine candidate antigenic epitopes and a multi-epitope peptide vaccine (NaeVac) design for the brain-eating amoeba Naegleria fowleri causing human meningitis

Naegleria fowleri, the brain-eating amoeba, is a free-living amoeboflagellate with three different life cycles (trophozoite, flagellated, and cyst) that lives in a variety of habitats around the world including warm freshwater and soil. It causes a disease called naegleriasis leading meningitis and primary amoebic meningoencephalitis (PAM) in humans. N. fowleri is transmitted through contaminated water sources such as insufficiently chlorinated swimming pool water or contaminated tap water, and swimmers are at risk. N. fowleri is found all over the world, and most infections were reported in both developed and developing countries with high mortality rates and serious clinical findings. Until now, there is no FDA approved vaccine and early diagnosis is urgent against this pathogen. In this study, by analyzing the N. fowleri vaccine candidate proteins (Mp2CL5, Nfa1, Nf314, proNP-A and proNP-B), it was aimed to discover diagnostic/vaccine candidate epitopes and to design a multi-epitope peptide vaccine against this pathogen. After the in silico evaluation, three prominent diagnostic/vaccine candidate epitopes (EAKDSK, LLPHIRILVY, and FYAKLLPHIRILVYS) with the highest antigenicities were discovered and a potentially highly immunogenic/antigenic multi-epitope peptide vaccine (NaeVac) was designed against the brain-eating amoeba N. fowleri causing human meningitis.

Addressing The Alarming Surge Of Naegleria And Other Water-Borne Diseases In Karachi: A Call For Urgent Action.

Naegleria fowleri, a thermophilic, free-living organism causes a life-threatening condition called primary amoebic meningoencephalitis (PAM). Naegleria thrives in warm freshwater but the rising number of cases in Karachi has raised concerns about the quality of the city’s domestic water supply. Pakistan has dealt with numerous healthcare burdens in the past, including outbreaks of diseases such as diphtheria, and cholera, and is persistently dealing with the challenge of polio. Moreover, bacterial contamination and improper water infrastructure pose a significant public health risk to several other water-borne diseases, prevalent in the city. Furthermore, the surge in cases of Naegleria adds to these ongoing health concerns. This brain-eating amoeba has the potential to spread beyond its current location, posing a significant threat to the nation's healthcare system. While the world is struggling to find an effective treatment for PAM, implementing preventive measures to improve water management, and surveillance can save lives and lessen the burden. The addition of chlorine to the water supply of the city by KWSB is a crucial step toward keeping this infection under control.

Unexpected trends of amoebic encephalitis in Pakistan.

Annual reported cases of Naegleria fowleri (NF), popularly known as brain eating amoeba, are becoming a huge challenge for Pakistani health authorities. Karachi has seen cases regularly up till the present but Lahore has not. The spread of this amoeba in non-chlorinated water is a major concern for the authorities. NF is an amoeba commonly found in warm freshwater environments such as lakes, hot springs and poorly chlorinated swimming pools. It poses a significant risk during hot weather when water-related recreational activities are popular. Where there is a non-chlorinated water supply, its spread is aggravated.

Clinical manifestations and outcome of patients with primary amoebic meningoencephalitis in Pakistan. A single-center experience.

Primary amoebic meningoencephalitis (PAM) is a rapidly progressing central nervous system (CNS) infection caused by Naegleria fowleri, a free-living amoeba found in warm freshwater. The disease progression is very rapid, and the outcome is nearly always fatal. We aim to describe the disease course in patients admitted with PAM in a tertiary care center in Karachi, Pakistan between the periods of 2010 to 2021. A total of 39 patients were included in the study, 33 males (84.6%). The median age of the patients was 34 years. The most frequent presenting complaint was fever, which was found in 37 patients (94.9%) followed by headache in 28 patients (71.8%), nausea and vomiting in 27 patients (69.2%), and seizures in 10 patients (25.6%). Overall, 39 patients underwent lumbar puncture, 27 patients (69.2%) had a positive motile trophozoites on CSF wet preparation microscopy, 18 patients (46.2%) had a positive culture, and 10 patients had a positive PCR. CSF analysis resembled bacterial meningitis with elevated white blood cell counts with predominantly neutrophils (median, 3000 [range, 1350-7500] cells/μL), low glucose levels median, 14 [range, 1-92] mg/dL), and elevated protein levels (median, 344 [range, 289-405] mg/dL). Imaging results were abnormal in approximately three-fourths of the patients which included cerebral edema (66.7%), hydrocephalus (25.6%), and cerebral infarctions (12.8%). Only one patient survived. PAM is a fatal illness with limited treatment success. Early diagnosis and prompt initiation of treatment can improve the survival of the patients and reduce mortality.

Multi-year variability of summer phytoplankton biomass and size structure in the northern Chukchi and East Siberian Seas, Arctic Ocean: role of light and nutrient availability

In the Arctic Ocean, the northern Chukchi and East Siberian Seas (NCESS) are vulnerable to climate change due to warming, sea ice melting, and surface freshening. To investigate how local physical forcing affects phytoplankton biomass and communities in this area, the multi-year (2015-2020) distributions of summer (August) chlorophyll-a concentrations and size structures using data collected by the Korea-Arctic Ocean Observing System (K-AOOS) program were investigated. The environmental characteristics and phytoplankton communities in the study area showed east-west regional differences. It is characterized by warm fresh waters and strong stratification to the east and cold saline waters and relatively high nutrient and sea ice concentrations to the west. Despite the differences between the east and west regions, patterns of the inter-annual variation in phytoplankton biomass and communities were similar across the entire study area, implying environmental controllers regulating phytoplankton in the NCESS. Inter-annually, higher sea surface salinity and weaker water column stratification were observed in 2017-2019 than in 2015 and 2020, implying the possibility of a potential modulation by the Arctic Oscillation. The shallower nitracline depth and higher surface nutrient concentrations since 2017 compared to the period from 2015-2016, indicate improved nutrient availability due to Atlantic water intrusion. However, average insolation has been relatively low since 2017, with the exception of August 2018. August mean phytoplankton biomass was highest in 2018 in the study area (average 83.7 mgm-2) and was dominated by large-sized phytoplankton. The low phytoplankton biomass in 2017 (23.9 mgm-2) and 2019 (62.4 mgm-2), despite similar characteristics of nutrient concentration to 2018, is likely due to lower average daily insolation in both years (206 μEm-2d-1 and 184 μEm-2d-1 in 2017 and 2019, respectively) compared to the 2018 average (271 μEm-2d-1). These results suggest that increased intrusion through upwelling of Atlantic water, along with nutrient loading, could be a pivotal driving factor contributing to this enhanced production in the NCESS, particularly under conditions where light levels are sufficient for phytoplankton growth and bloom development.

Distinct vertical behavior of key Arctic copepods following the midnight sun period in the East Siberian continental margin region, Arctic Ocean

Diel vertical migration (DVM) of zooplankton plays a vital role in biological carbon pump and food web interactions. However, there is considerable debate about the DVM of zooplankton in response to environmental changes in the Arctic Ocean. We investigated DVM behavior in the key Arctic copepods Calanus glacialis, Calanus hyperboreus, and Metridia longa following the midnight sun period in the East Siberian continental margin region. The two Calanus species showed non-DVM behaviors, whereas M. longa showed a typical DVM pattern consistent with the solar radiation cycle. Additionally, these species showed different vertical distributions. Calanus glacialis was distributed at depths above 20 m in the warm fresh water, where the highest density gradient was observed. Calanus hyperboreus was distributed at depths between 30 and 55 m in the cold salty water, where a high contribution of micro phytoplankton and the subsurface chlorophyll maximum (SCM) layer were observed. M. longa was found across a broader range of temperature and salinity than both Calanus species, and it was distributed in the upper water column, where the SCM layer was observed at night and at depths between 100 and 135 m in the daytime. These results imply that M. longa can be well adapted to the changing Arctic Ocean environment, where sea ice loss and ocean warming are ongoing, whereas C. hyperboreus can be the most vulnerable to these changes. These findings provide important information for understanding variations in the vertical distributions of key copepod species in the rapidly changing Arctic marine environment.

Approaches for reducing the iodine content of the brown seaweed Saccharina latissima—effects on sensory properties

AbstractThe effects of steam- or warm water treatments of the brown seaweed Saccharina latissima on iodine content and nutrient and sensory profiles were investigated. Warm freshwater or seawater treatments reduced the dry weight iodine content by 73% and 59%, respectively. Steam treatment was less efficient and only reduced the iodine content by 26%. Freshwater treatment resulted in a greater reduction in total solids and loss of water-soluble components, mainly reducing the content of ash and carbohydrates. Seawater treatment had a small effect on total solid content but altered the mineral profile leading to an increase in sodium and decrease in potassium content. The sensory profiles reflected the observed differences in composition as it revealed a significantly higher saltiness of the seawater-treated sample compared to other treatments, as well as a higher umami intensity compared to the freshwater-treated sample. The latter was characterized by low scores across all sensory attributes, indicating greater loss of flavour-active compounds. Further, including seawater-treated S. latissima at relatively low levels (0.5% and 1%) significantly altered the flavour of a commercial spinach soup compared to the same preparation without seaweed. Therefore, warm water treatment using seawater could be an alternative method for lowering the iodine content in brown seaweed while limiting nutrient loss and maintaining its flavour potential for applications in the food industry.

Evaluation of the fish acute toxicity test for pesticide registration

The U.S. Environmental Protection Agency (USEPA) uses the in vivo fish acute toxicity test to assess potential risk of substances to non-target aquatic vertebrates. The test is typically conducted on a cold and a warm freshwater species and a saltwater species for a conventional pesticide registration, potentially requiring upwards of 200 or more fish. A retrospective data evaluation was conducted to explore the potential for using fewer fish species to support conventional pesticide risk assessments. Lethal concentration 50% (LC50) values and experimental details were extracted and curated from 718 studies on fish acute toxicity submitted to USEPA. The LC50 data were analysed to determine, when possible, the relative sensitivity of the tested species to each pesticide. One of the tested freshwater species was most sensitive in 85% of those cases. The tested cold freshwater species was the most sensitive overall among cases with established relative sensitivity and was within 3X of the LC50 value of the most sensitive species tested in 98% of those cases. The results support potentially using fewer than three fish species to conduct ecological risk assessments for the registration of conventional pesticides.

Laboratory Diagnosis of Primary Amoebic Meningoencephalitis.

Primary amebic meningoencephalitis (PAM) is a fulminant fatal human disease caused by the free-living amoeba Naegleria fowleri. Infection occurs after inhalation of water containing the amoeba, typically after swimming in bodies of warm freshwater. N.fowleri migrates to the brain where it incites meningoencephalitis and cerebral edema leading to death of the patient 7 to 10days postinfection. Although the disease is rare, it is almost always fatal and believed to be underreported. The incidence of PAM in countries other than the United States is unclear and possibly on track to being an emerging disease. Poor prognosis is caused by rapid progression, suboptimal treatment, and underdiagnosis. As diagnosis is often performed postmortem and testing is only performed by a few laboratories, more accessible testing is necessary. This article reviews the current methods used in the screening and confirmation of PAM and makes recommendations for improved diagnostic practices and awareness.

Formation of oil-particle aggregates with motor oil and kaolinite clay in cold and warm freshwater.

Motor oil is one of the most common pollutants in stormwater runoff in freshwater ecosystems. It can form aggregates with solids (creating oil particle aggregates, OPAs) which complicates the understanding of the fate and transport of motor oil, particularly in cold freshwater, conditions that have not been studied extensively. Laboratory and numerical experiments were conducted with kaolinite clay and three types of motor oil in both cold and warm freshwater, in which: (1) the interaction of clay particles with motor oil was experimentally investigated, in response to changes in oil viscosity, water temperature, and mixing intensity; (2) variability in particle size distribution of the formed OPAs was measured; and (3) a mechanistic OPA formation model was applied and results were compared with experimental data. The results showed that kaolinite clay and motor oil formed mostly droplet-type OPAs, lower-viscosity oil tended to form a wider size range of OPAs, and higher mixing intensity and higher water temperature produced larger numbers of smaller OPAs. Although there was a reasonably good match between the experimental data and the modeling results, more research is needed to further improve the modeling framework.

Effects of formaldehyde ( Parasite‐S ®) on biofilter nitrification from a cold and a warm freshwater RAS

The effect of Parasite-S® (an aqueous formaldehyde solution) on the nitrification processes of biofilters was evaluated in two recirculating aquaculture systems (RASs). Rearing tanks in the warmwater RAS contained yellow perch (Perca flavescens) and grass carp (Ctenopharyngodon idella) with an initial weight of 166.8 kg and a mean density of 39.5 kg/m3. Rearing tanks in the coldwater RAS contained rainbow trout (Oncorhynchus mykiss) and lake trout (Salvelinus namaycush) with an initial weight of 1377.8 kg at a system density of 41.9 kg/m3. Parasite-S® was administered to the entire system on four consecutive days in both trials to achieve a nominal concentration of 14.8 mg/L formaldehyde (40 mg/L formalin) at the biofilter. Removal efficiencies for total ammonia nitrogen (TAN) and nitrite nitrogen were measured as indicators of biofilter nitrification processes. The active ingredient in Parasite-S®, formaldehyde, was measured until it was below the method detection limit of 0.8 mg/L. TAN volumetric removal rate was significantly decreased in both systems after formaldehyde addition and remained below pre-exposure efficiency in the coldwater RAS. Nitrite nitrogen volumetric removal rate was not significantly different, but the slope and intercepts were less after formaldehyde addition indicating an effect on the nitrifying bacteria. Although removal rates were decreased, no mortality occurred after four consecutive formaldehyde indefinite bath exposures in either system.

Projected future changes in the contribution of Indo-Pacific sea surface height variability to the Indonesian throughflow

The Indonesian throughflow (ITF) transports a significant amount of warm freshwater from the Pacific to the Indian Ocean, making it critical to the global climate system. This study examines decadal ITF variations using ocean reanalysis data as well as climate model simulations from the Coupled Model Inter-comparison Project Phase 5 (CMIP5). While the observed annual cycle of ITF transport is known to be correlated with the annual cycle of sea surface height (SSH) difference between the Pacific and Indian Oceans, ocean reanalysis data (1959–2015) show that the Pacific Ocean SSH variability controls more than 85% of ITF variation on decadal timescales. In contrast, the Indian Ocean SSH variability contributes less than 15%. While those observed contributions are mostly reproduced in the CMIP5 historical simulations, an analysis of future climate projections shows a 25–30% increase in the Indian Ocean SSH variability to decadal ITF variations and a corresponding decrease in the Pacific contribution. These projected changes in the Indian Ocean SSH variability are associated with a 23% increase in the amplitudes of negative zonal wind stress anomalies over the equatorial Indian Ocean, along with a 12º eastward shift in the center of action in these anomalies. This combined effect of the increased amplitude and eastward shift in the zonal wind stress increases the SSHA variance over the Indian Ocean, increasing its contribution to the ITF variation. The decadal ITF changes discussed in this study will be crucial in understanding the future global climate variability, strongly coupled to Indo-Pacific interactions.

Differential Growth Rates and In Vitro Drug Susceptibility to Currently Used Drugs for Multiple Isolates of Naegleria fowleri.

ABSTRACTThe free-living amoeba Naegleria fowleri, which typically dwells within warm, freshwater environments, can opportunistically cause primary amoebic meningoencephalitis (PAM), a disease with a mortality rate of >97%. The lack of positive treatment outcomes for PAM has prompted the discovery and development of more effective therapeutics, yet most studies utilize only one or two clinical isolates. The inability to assess possible heterogenic responses to drugs among isolates from various geographical regions hinders progress in the discovery of more effective drugs. Here, we conducted drug efficacy and growth rate determinations for 11 different clinical isolates by applying a previously developed CellTiter-Glo 2.0 screening technique and flow cytometry. We found significant differences in the susceptibilities of these isolates to 7 of 8 drugs tested, all of which make up the cocktail that is recommended to physicians by the U.S. Centers for Disease Control and Prevention. We also discovered significant variances in growth rates among isolates, which draws attention to the differences among the amoeba isolates collected from different patients. Our results demonstrate the need for additional clinical isolates of various genotypes in drug assays and highlight the necessity for more targeted therapeutics with universal efficacy across N. fowleri isolates. Our data establish a needed baseline for drug susceptibility among clinical isolates and provide a segue for future combination therapy studies as well as research related to phenotypic or genetic differences that could shed light on mechanisms of action or predispositions to specific drugs.IMPORTANCE Naegleria fowleri, also known as the brain-eating amoeba, is ubiquitous in warm freshwater and is an opportunistic pathogen that causes primary amoebic meningoencephalitis. Although few cases are described each year, the disease has a case fatality rate of >97%. In most laboratory studies of this organism, only one or two well-adapted lab strains are used; therefore, there is a lack of data to discern if there are major differences in potency of currently used drugs for multiple strains and genotypes of the amoeba. In this study, we found significant differences in the susceptibilities of 11 N. fowleri isolates to 7 of the 8 drugs currently used to treat the disease. The data from this study provide a baseline of drug susceptibility among clinical isolates and suggest that new drugs should be tested on a larger number of isolates in the future.

Waters of death: Naegleria Fowleri (the brain eating Amoeba).

Dear Madam, with the commencement of the monsoon season and temperature on the rise again, the incidence of Naegleria Fowleri (Brain-eating amoeba) is also on the rise. The death toll has risen to six in Karachi so far this year as it claims lives of a 19-year-old teenager and an 8-year-old boy adding to the previous 4 deaths out of which one was a 30-year-old neurosurgeon and an another 8-year-old boy. (1) (2) All of them had acquired this amoeba through contaminated water. A recent Karachi water and sewage board (KWSB) report disclose that 70% of its pumping stations are supplying water containing either no or low Chlorine, which is a major threat to the residents of Karachi (3). Naegleria Fowleri is an amoeba and causes a devastating brain infection known as primary amoebic meningoencephalitis (PAM). The amoeba is commonly found in warm freshwaters. It infects people when contaminated water enters the body through the nose while performing ablution (Wudu), swimming or bathing, and from there, it travels to the brain (4). (PAM) destroys the brain tissue, and its symptoms start in about five days including headache, fever, nausea & vomiting initially. The later symptoms include stiff neck, altered mental status, seizures, hallucinations and can rapidly progress to death (4). The recommended treatment for Naegleria include a combination of drugs including Amphotericin B (Anti-Fungal) and Miltefosine; however, few people survive, even with treatment (5). (PAM) is difficult to detect because the disease progresses so rapidly that the diagnosis is usually made after death. This is not the first time. Naegleria claimed five lives back in 2016 and many more previously (6), which raises a serious question on the efficiency and preventive measures taken by the concerned departments. The fact that the temperature in Karachi remains warm for the most part of the year and on top of the contaminated water supply (less chlorinated), is gravely concerning. Since prevention is better than cure, the general public must be made aware of Naegleria and advised to avoid swimming, bathing or performing ablution in unchlorinated water. Also, before the summer starts, people should get their underground and overhead water tanks cleaned and chlorinated. Along with warning people, measures should be taken by the responsible departments to make water safe for the citizens and prevent any further deaths by this lethal amoeba.

Naegleria fowleri from Pakistan Has Type-2 Genotype.

Background:Primary amoebic meningoencephalitis (PAM) is an acute and fulminant CNS infection caused by Naegleria fowleri. Recreational activities and ritual ablution with contaminated warm fresh water are the main reason of PAM. Pakistan ranked the second most affected country, where most of the PAM incidences were reported from Karachi, Pakistan.Methods:In May, 2019, a 28-yr-old suspected PAM patient came to the Imam Zain-Ul-Abdin Hospital, Karachi. Biochemical and cytological investigations of patient‘s CSF were carried out at Karachi Diagnostic Center and Molecular Biology Lab. Sequencing of Naegleria sp. specific (ITS) primer-based amplicons was performed from both patient‘s CSF and water samples followed by multiple sequence alignment and phylogenetic studies.Results:Biochemical and cytological investigations of patient‘s CSF showed 5 mg/dl glucose, 240 mg/dl total protein and 2260/mm3 TLC suggesting acute meningoencephalitis. PCR-based analyses of patient‘s CSF and his residential tap water samples using Naegleria sp. specific (ITS) and N. fowleri specific primers revealed the presence of N. fowleri DNA. Nucleotide sequences of ITS primer-based amplicons from both patient‘s CSF and water samples were submitted in GenBank under the accession numbers MT726981.1 and MT726226.1, respectively. According to phylogenetic analysis, N. fowleri isolate from Pakistan has shown the least node age of seven.Conclusion: Here, for the very first time in Pakistan, N. fowleri genotype has been identified as type-2. Phylogenetic analysis showed that N. fowleri isolate from Pakistan is among the latest descendants, i.e., evolved later in life.

EdU Incorporation To Assess Cell Proliferation and Drug Susceptibility in Naegleria fowleri.

Naegleria fowleri is a pathogenic free-living amoeba that is commonly found in warm freshwater and can cause a rapidly fulminant disease known as primary amoebic meningoencephalitis (PAM). New drugs are urgently needed to treat PAM, as the fatality rate is >97%. Until recently, few advances have been made in the discovery of new drugs for N. fowleri, and one drawback is the lack of validated tools and methods to enhance drug discovery and diagnostics research. In this study, we aimed to validate alternative methods to assess cell proliferation that are commonly used for other cell types and develop a novel drug screening assay to evaluate drug efficacy on N. fowleri replication. EdU (5-ethynyl-2'-deoxyuridine) is a pyrimidine analog of thymidine that can be used as a quantitative endpoint for cell proliferation. EdU incorporation is detected via a copper catalyzed click reaction with an Alexa Fluor-linked azide. EdU incorporation in replicating N. fowleri was validated using fluorescence microscopy, and quantitative methods for assessing EdU incorporation were developed by using an imaging flow cytometer. Currently used PAM therapeutics inhibited N. fowleri replication and EdU incorporation in vitro. EdA (7-deaza-2'-deoxy-7-ethynyladenosine), an adenine analog, also was incorporated by N. fowleri but was more cytotoxic than EdU. In summary, EdU incorporation could be used as a complimentary method for drug discovery for these neglected pathogens.

Copper detoxification machinery of the brain-eating amoeba Naegleria fowleri involves copper-translocating ATPase and the antioxidant system

Copper is a trace metal that is necessary for all organisms but toxic when present in excess. Different mechanisms to avoid copper toxicity have been reported to date in pathogenic organisms such as Cryptococcus neoformans and Candida albicans. However, little if anything is known about pathogenic protozoans despite their importance in human and veterinary medicine. Naegleria fowleri is a free-living amoeba that occurs naturally in warm fresh water and can cause a rapid and deadly brain infection called primary amoebic meningoencephalitis (PAM). Here, we describe the mechanisms employed by N. fowleri to tolerate high copper concentrations, which include various strategies such as copper efflux mediated by a copper-translocating ATPase and upregulation of the expression of antioxidant enzymes and obscure hemerythrin-like and protoglobin-like proteins. The combination of different mechanisms efficiently protects the cell and ensures its high copper tolerance, which can be advantageous both in the natural environment and in the host. Nevertheless, we demonstrate that copper ionophores are potent antiamoebic agents; thus, copper metabolism may be considered a therapeutic target.