Primary+Amoebic+Meningoencephalitis

Primary amoebic meningoencephalitis (PAM) is an infection of the central nervous system (CNS) caused by a free-living amoeba known as //[|Naegleria fowleri]//. [|N. fowleri], a thermophilic amoeboflagellate, is normally found in warm freshwater bodies such as lakes, rivers, hot springs, and other thermally polluted waters. The infection is contracted when contaminated water is forcibly drawn into the nasal cavity.[| //N. fowleri//] adheres to the nasal mucosa, and subsequently migrates to the [|olfactory bulbs], using the olfactory nerves for guidance. Once inside the brain, //[|N. fowleri]// proliferates and initiates cytotoxic effects that cause inflammation and necrotic hemorrhaging. Symptoms typically appear within 2-15 days after exposure. The symptoms include headache, fever, stiff neck, vomiting, compromised neurological and mental functions, seizures, and coma. The disease is rapidly progressive and highly fatal. Death typically occurs within 3-7 days after the first sign of symptoms** [2] **.

 Only 235 cases of PAM have been reported worldwide since it was discovered in 1968. Despite its rare occurrence, medical professionals currently face many challenges with the disease. Awareness is low among clinicians, diagnosis is difficult and time consuming, and treatment is unsuccessful in most cases. In addition, millions of people are exposed to the amoeba annually; however, no reliable estimation of occurrence of PAM exists. Current research primarily investigates novel therapies, diagnostic assays, and the underlying pathogenesis of the disease** [2] .**

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=**1.1 Pathogenesis** =

1.1a Cause: //Naegleria fowleri//
=media type="custom" key="13851726" align="right" = Primary amoebic meningoencephalitis (PAM) is a rare and rapidly fatal infection of the human central nervous system (CNS) caused by a free-living protist called //Naegleria fowleri//. The //Naegleria// genus belongs to a family collectively known as [|Vahlkamphfidae] ** [4] **. Although more than 40 species of //Naegleria// have been identified, //N. fowleri// is the only pathogenic species associated with a human infection. //N. fowleri// is a thermophilic amoeba capable of surviving and proliferating in temperatures as high as 45 degrees Celsius. Its geographic distribution throughout the world is ubiquitous. //N. fowleri// has been found in warm freshwater of tropical, subtropical, and temperate environments. It is naturally found in lakes, rivers, ponds, hot springs, puddles, and soil. In addition, it is also found in tap water, baths, swimming pools, sewage canals and in the effluents of power plants because of thermal pollution and poor maintenance. It survives by feeding on bacteria** [4] **.

1.1b Life Cycle
 //N. fowleri// is a triphasic amoeboflagellate. Its life cycle consists of 3 distinct morphologically and functional states. Its existing state depends on its environment.

=== === **Cyst Stage** <span style="font-family: Arial,Helvetica,sans-serif;"> <span style="font-family: Arial,Helvetica,sans-serif;">The cyst is the resistant morphological state of //N. fowleri// that occurs under hostile environmental conditions. Although nonpathogenic in this stage, cysts may also enter the nasal passageway, where they can then convert to trophozoites and induce infection ** [5,7] **<span style="font-family: Arial,Helvetica,sans-serif;">.

<span style="font-family: Arial,Helvetica,sans-serif;"> **Trophozoite Stage** <span style="font-family: Arial,Helvetica,sans-serif;"> The[| trophozoite] is the pathogenic form of //N. fowleri// for humans. In this state it is capable of proliferation and feeding. Proliferation occurs through binary fission. A 'food-cup' structure, called an amoebastome, develops on the surface of the amoeba for feeding. The elongated, slug-like trophozoite is typically 10-25 µ m in size and is capable of locomotion by means of pseudopodia <span style="font-family: Arial,Helvetica,sans-serif;"> ** [5,7] **.

<span style="font-family: Arial,Helvetica,sans-serif;"> **Flagellate Stage** <span style="font-family: Arial,Helvetica,sans-serif;"> <span style="font-family: Arial,Helvetica,sans-serif;">The [|flagellate] is a transient morphological state of //N. fowleri// that occurs when deprived of nutrients in water. The characteristic feature of //N. fowleri// during this stage is a pear-shaped structure. //N. fowleri// is nonpathogenic in this stage ** [5,7] **<span style="font-family: Arial,Helvetica,sans-serif;">.

<span style="font-family: Arial,Helvetica,sans-serif;">1.1c Infection
<span style="font-family: Arial,Helvetica,sans-serif;"> Infection occurs when the trophozoite adheres to the nasal mucosa after contaminated water enters the nasal cavity. The adherence of the trophozoite to neurons and other cell types involves binding to extracellular matrix glycoproteins such as[| fibronectin], [|collagen], and [|laminin]; at least in vitro [7]. For example, Han et al, identified a fibronectin binding surface protein in extracts of //N. fowleri// using binding assays. Once adhered to the nasal mucosa, the trophozoite migrates to the olfactory bulbs by advancing through the [|cribriform plate]; using the olfactory nerves for guidance** [2] **<span style="font-family: Arial,Helvetica,sans-serif;">. Proliferation of the trophozoite begins in the olfactory bulbs, followed by host-cell interactions that trigger an inflammatory response and necrosis. Once inside the brain, //N. fowleri// proliferates and initiates cytotoxic effects that cause inflammation and necrotic hemorrhaging. The amoebastomes, pseudopodial food-cup projections, mediate the host-tissue invasion and ingestion. NFa1 is thought to be the key protein that mediates the ability of //N. fowleri// for adherence, locomotion, and the formation of food-cups for ingestion. Shin //et al// showed that NFa1 was abundantly expressed and localized around the pseudopodial projections and food-cups** [10] **<span style="font-family: Arial,Helvetica,sans-serif;">.

<span style="font-family: Arial,Helvetica,sans-serif;"> Entry through the nasal cavity is predominantly the main form of transmission; however, in some cases //N. fowleri// can enter through the ear when the [|tympanic membrane] is ruptured. In addition, PAM may be contracted by dust containing //N. fowleri//. The infection is contracted when contaminated water is forcibly drawn into the nasal cavity. <span style="font-family: Arial,Helvetica,sans-serif;"> ** [4,5] **<span style="font-family: Arial,Helvetica,sans-serif;">.

=<span style="font-family: Arial,Helvetica,sans-serif;">**1.2 Clinical Manifestation and Symptoms** =

<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-family: Arial,Helvetica,sans-serif;">PAM is a progressive form of [|meningoencephalitis] with a rapid onset. Symptoms typically become apparent 2-15 days after exposure. However, in some cases the onset of illness can be within 24 hours ** [2] **<span style="font-family: Arial,Helvetica,sans-serif;">. <span style="font-family: Arial,Helvetica,sans-serif;"> <span style="font-family: Arial,Helvetica,sans-serif;">. The clinical symptoms of PAM are similar to those seen in viral or bacterial meningitis. In fact, PAM is very difficult to diagnose because it does not have any distinct clinical features of its own [3]. Symptoms that occur early on in the disease include localized headaches in the frontal and temporal regions of the head, fever, vomiting, rhinitis, and a stiff neck ** [5] **<span style="font-family: Arial,Helvetica,sans-serif;">. The symptoms become more severe as the disease progresses. For instance, a patient will present with neurological abnormalities, a comprised mental state, [|diplopia], [|photophobia], seizures, and coma. The patient typically dies within one week after the onset of symptoms. Early diagnosis and treatment are critical for survival ** [2] **<span style="font-family: Arial,Helvetica,sans-serif;">.

<span style="font-family: Arial,Helvetica,sans-serif;">

=<span style="font-family: Arial,Helvetica,sans-serif;">**1.3 Diagnosis** =

<span style="font-family: Arial,Helvetica,sans-serif;">The current methods for diagnosing PAM are based on the identification of //N. fowleri// in clinical samples.

<span style="font-family: Arial,Helvetica,sans-serif;">1.3a Histological and Morphological Assays
<span style="font-family: Arial,Helvetica,sans-serif;"> **Wet-mounted Microscopy**

<span style="font-family: Arial,Helvetica,sans-serif;"> Wet-mounted microscopy is used to examine the cerebrospinal fluid (CSF) for the presence of trophozoites.The technique involves phase-contrast microscopy and the ability to identify the morphological and locomotive characteristics of the trophozoite. A typical trophozoite is about 10-25 µm in size with a large nucleolus located in its center. The pattern of locomotion is usually rapid and directional, involving the use of [|pseudopodia]** [2] **<span style="font-family: Arial,Helvetica,sans-serif;">. A drawback to this technique is the physician's inability to identify the trophozoite, because it is often mistaken for a[| leukocyte] ** [5] **<span style="font-family: Arial,Helvetica,sans-serif;">.

<span style="font-family: Arial,Helvetica,sans-serif;"> **Flagellation Test (FT)**

<span style="font-family: Arial,Helvetica,sans-serif;"> The flagellation test is a supplementary diagnostic tool used for detecting //N. fowleri//. The trophozoites are placed in a hypotonic solution and examined to see whether they convert to the flagellate state. The test is conducted a second time using a higher temperature to differentiate between different species of //Naegleria//. The drawback to this technique is an incidence for false negative results. Consequently, an additional tool is used regardless of the outcome. For instance, [|polymerase chain reaction] ([|PCR]), ELISA, and[| restriction length fragment polymorphism] ([|RFLP]).

<span style="font-family: Arial,Helvetica,sans-serif;"> **Cell Culture**

<span style="font-family: Arial,Helvetica,sans-serif;"> <span style="font-family: Arial,Helvetica,sans-serif;"> Cell culture examination is subsequently used if //N. fowleri// is suspected in wet-mount microscopy. The procedure for this test involves incubating a small drop of sedimented CSF after being mixed with 1mL of sterile water and placed in a tube. The incubation lasts for about 3 hours at 37 degrees Celsius. The physician must examine the culture regularly for the presence of actively mobile pear-shaped flagellates; typically composed of two terminal flagella. In addition, the CSF is typically grayish or yellowish-white during the early phase of infection. As the disease progresses the CSF turns red as the amount of red blood cells increases ** [4,5] **<span style="font-family: Arial,Helvetica,sans-serif;">.

<span style="font-family: Arial,Helvetica,sans-serif;">1.3b Immunological Assays
<span style="font-family: Arial,Helvetica,sans-serif;"> **Enzyme-linked Immunosorbent Assay (ELISA)**

<span style="font-family: Arial,Helvetica,sans-serif;"> <span style="font-family: Arial,Helvetica,sans-serif;"> Several immunological assays have been developed to identify //N. fowleri//. These methods include [|immunoelectrophoresis] and indirect [|immunofluorescence staining]. Recently, the [|enzyme-linked immunosorbent assay](ELISA) was adopted for the detection of N. fowleri using a specific [|monoclonal antibody] (Mab 5D12) ** [8] **<span style="font-family: Arial,Helvetica,sans-serif;">. [|ELISA] is capable of distinguishing //N. fowleri// from other species of Naegleria and detecting it in all three stage of its lifecycle. The drawback is the requirement of a cell culture before [|ELISA] can be performed.

<span style="font-family: Arial,Helvetica,sans-serif;">1.3c Biochemical and Molecular Assays
<span style="font-family: Arial,Helvetica,sans-serif;"> **Polymerase Chain Reaction (PCR) Assay**

<span style="font-family: Arial,Helvetica,sans-serif;"> Polymerase chain reaction protocol are currently the best diagnostic tools for PAM. The protocols are comprised of conventional, [|nested], [|multiplex], and [|real-time PCR] methods. Recently, Obernauerova et al developed a novel addition to the real-time PCR diagnostic protocol that allows for fast and accurate detection of //N. fowleri//. The addition is a fluorescent labeling probe that selectively targets a gene unique to //N. fowleri// called Mp2Cl5.

<span style="font-family: Arial,Helvetica,sans-serif;"> 1.3d Issues with Diagnosing PAM
<span style="font-family: Arial,Helvetica,sans-serif;"> There are 3 main issues that make the diagnosis of PAM rather difficult. Patients that contract PAM do not have any distinct features that differentiate the infection from other forms of viral or bacterial meningitis. In addition, awareness among clinicians is low because the disease is very rare. Lastly, the disease is highly fatal with a rapid onset. As a result, the right diagnosis is often too late for effective treatment.

=<span style="font-family: Arial,Helvetica,sans-serif;">**1.4 Pathophysiology** =

<span style="font-family: Arial,Helvetica,sans-serif;">1.4a Inflammation and Purulent Exudate
<span style="font-family: Arial,Helvetica,sans-serif;"> Inflammation is one of three main features that underlie the pathophysiology of PAM. Inflammation initiates in the [|olfactory bulbs], and eventually spreads to other areas of the brain and meninges. According to one study, the inflammation is limited only to the frontal regions of the brain. The current understanding regarding the //N. fowleri-//induced inflammatory process initially involves two types of cell-cell interactions. The first involves //N. fowleri//-astroglial cell interactions and the activation of[| Interleukin-8] ([|IL-8]) genes through the[| ERK-1/2 signaling pathway]. //N. fowleri// stimulates the activity of (1) [|extracellular signal-regulated kinases] (ERKs) and the (2) DNA binding activity of [|activator protein-1] (AP-1) to upregulate the expression of [|IL-8] genes in human[| astroglial cells]. The[| IL-8] genes produce [|cytokines] that play a key role in the development of the inflammatory response against the trophozoite. //N. Fowler-microglial// cell interactions are also implicated in the process of inflammation. Pro-inflammatory [|cytokines], such as [|interleukin-6], [|IL1-beta], and[| tumor necrosis factor-alpha], are released when //N. fowleri// interacts with [|microglia]** [3] **<span style="font-family: Arial,Helvetica,sans-serif;">. Purulent exudate is typically associated with the inflammatory process. The exudate accumulates within the base of the brain, olfactory bulbs, brainstem, cerebellum, and in between sulci, causing congestion and [|hyperemia]

<span style="font-family: Arial,Helvetica,sans-serif;">media type="custom" key="13864326" align="right"
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-family: Arial,Helvetica,sans-serif;">in the [|leptomeninges] ** [2] **<span style="font-family: Arial,Helvetica,sans-serif;">.

<span style="font-family: Arial,Helvetica,sans-serif;">1.4b Brain Hemorrhaging and Necrosis
<span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-family: Arial,Helvetica,sans-serif;"> The hemorrhagic and necrotic events are mediated by //N. fowleri//-host cell interactions. A series of studies examining the interaction between //N. fowleri// and mammalian cells have shown that various proteins are involved.The trophozoite induces necrotic hemorrhaging by releasing cytolytic proteins and by [|trogocytosis]. These mechanisms are used to destroy neurons and other cell types for easy digestion. The mechanism selected by the trophozoite depends on its pathogenic strength, at least in vitro. For example, weakly pathogenic trophozoites use [|trogocytosis], a mechanism involving the ingestion of mammalian cells, such as neurons, with a 'food-cup' structure located on the surface of the trophozoite. In contrast, highly pathogenic trophozoites consume mammalian cells after releasing cytolytic proteins ** [8] **<span style="font-family: Arial,Helvetica,sans-serif;">.

<span style="font-family: Arial,Helvetica,sans-serif;"> <span style="font-family: Arial,Helvetica,sans-serif;"> //N. fowleri// secretes enzymes that degrade a wide variety of connective tissue and structural proteins. For instance, //N. Fowleri// degrades [|sphingomyelin] by releasing [|proteases], [|acid hydrolases], [|phospholipases], and phospholipolytic enzymes. In addition, the trophozoite secretes [|neuraminidases] and [|elastase]. These enzymes are responsible for degrading collagen and [|proteoglycans], and for altering glycolipid and phospholipid composition to induce demyelination, respectively. Since the cytopathology of PAM is poorly understood, the proteins aforementioned are by no means an exhaustive list and the exact determinants of the pathogenicity remain unclear ** [8] **<span style="font-family: Arial,Helvetica,sans-serif;">.

<span style="font-family: Arial,Helvetica,sans-serif;"> Neuronal lysis by the trophozoite triggers an inflammatory response, which ultimately leads to the destruction of brain tissue. The inflammatory response is associated with necrotic hemorrhaging, resulting in widespread lesions through the cortex and spinal cord. The areas of the CNS most susceptible to the hemorrhaging include the base of the brain, olfactory bulbs, temporal and orbitofrontal lobes, hypothalamus, brainstem, and cervical portion of the spinal cord <span style="font-family: Arial,Helvetica,sans-serif;"> ** [2] **<span style="font-family: Arial,Helvetica,sans-serif;">.

=<span style="font-family: Arial,Helvetica,sans-serif;">**1.5 Treatment** =

<span style="font-family: Arial,Helvetica,sans-serif;">1.5a Amphotericin B (AMB) Therapies
<span style="font-family: Arial,Helvetica,sans-serif;"> <span style="font-family: Arial,Helvetica,sans-serif;"><span style="font-family: Arial,Helvetica,sans-serif;"> There is no cure for PAM at present (shin et al). The primary therapeutic agent for PAM typically involves a polyene antifungal drug called [|amphotericin B] ([|AMB]). It is administered alone or in conjugation with other drugs [3]. For instance, AMB has been administered with [|imidazole] and [|rifampicin] drugs, such as [|miconazole], [|fluconazole], and [|ornidazole] ** [2,5]  **<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12px;">. Treatment is usually limited to 10 days because of adverse side effects, such as electrolyte imbalances, kidney damage, and hematopoietic changes that result in [|anemia] ** [2] **<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12px;">. Consequently, many agents, including antifungal, antiprotozoal, antibacterial, and antipsychotic drugs, have been screened for therapeutic activity against //N. fowleri//; in vivo and in vitro ** [16]  .**

<span style="font-family: Arial,Helvetica,sans-serif;">1.5b Novel Research in Therapeutic Strategies
<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;"> In 2002, Brenner //et al// conducted a series of studies to test the amoebicidal activity of a [|macrolide] antibiotic, called [|azithromycin], against //N. fowleri// in a mouse model of PAM and in vitro. The study concluded that azithromycin was highly active against //N. fowleri//. An additional study in 2007 investigated the effect of combining [|AMB] and [|azithromycin] in vitro and in mouse models of PAM. A synergistic effect was produced against //N. fowleri// when [|AMB] and [|azithromycin] were used in conjunction. [|Azithromycin] is believed to suppress the synthesis of bacterial proteins and inhibit translocation by binding to the 50S ribosomal subunit. However, the exact mechanism of action against //N. fowleri// is not clear.

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;"> A recent study conducted by Shin //et al//, also investigated the efficacy of [|miltefosine] and [|chlorpromazine] against //N. fowleri// in both in vivo and in vitro models. [|Chlorpromazine] and miltefosine both demonstrated amoebicidal activity and repressed the proliferation of //N. fowleri// in vitro//.// [|Chlorpromazine] had the highest clinical efficacy against //N. fowleri// infection in vivo. A comparative analysis showed that chlorpromazine was less toxic and more effective than AMB when treating for infection both in vivo and in vitro

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;"> The Nfa1 gene, responsible for mediating the pathogenicity of //N. fowleri//, is another promising target for developing a novel therapeutic strategy. [|Cytochalasin D], an actin polymerization inhibitor, was shown to be effective in treating against //N. fowleri// by reducing its ability to form an amoebastome in vitro. In addition, an antisense oligonucleotide of the Nfa1 gene and an anti-Nfa1 polyclonal antibody produced the same effects. Most recently, [|RNA interference] ([|RNAi]) was used to down-regulate the levels of Nfa1 mRNA and protein. This resulted in a lowered cytotoxic effect on macrophages in vitro.

=<span style="font-family: Arial,Helvetica,sans-serif;">**1.6 Epidemiology** =

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;"><span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;">In 1965, M. Fowler and R.F. Carter identified and described //N. fowleri// as a pathogenic species causing infection in humans ** [2,4] **<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;">. Since then, only 235 cases of PAM have been reported worldwide in over 16 countries, making the disease rare ** [4] **<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;">. In over 95% of cases, the individual has died due to the severe complications caused by inflammation and hemorrhagic necrosis. The disease affects young healthy individuals with recent exposure to water contaminated with //N. fowleri//. The estimated occurrence for PAM is unreliable, however, the incidence of PAM is highest in young males; possibly due to a higher rate of males engaging in recreational water-based activities. ** [2,16] **<span style="font-family: Arial,Helvetica,sans-serif;">.

<span style="font-family: Arial,Helvetica,sans-serif;">1.6a Worldwide Occurrence
<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;"> **Cases in Africa** <span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;"> <span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;">Only six cases of PAM were reported in Africa: four in Nigeria and one in both Namibia and Madagascar. The cases in Nigeria were particularly unique because it was hypothesized that three of four patients contracted the infection from dust containing cysts ** [4] **<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;">.

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;"> **Cases in Asia** <span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;"> <span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;">In Asia there have been 39 confirmed cases of PAM: 17 patients in Pakistan, 12 in Thailand, 7 in Indian, two in Japan, and one in China. In Pakistan the disease was contracted from the use of unfiltered tap water during a 17-month period. In Thailand, India, and China the disease was contracted from swimming in lakes, canals, hot springs, or thermally polluted waters. In Japan, the source of infection is unknown ** [4] **<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;">.

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;"> **Cases in Australia** <span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;"> <span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;"> The 19 reported cases of PAM in Australia occurred in a town located in the Southern state. The town's water supply was contaminated with //N. fowleri//. In New Zealand, nine patients contracted the disease after swimming in geothermal water ** [2] **<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;">.

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;"> **Cases in Europe** <span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;"> <span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;"> There have been 24 confirmed cases of PAM reported in Europe: specifically in the Czech Republic, Belgium, Italy, and the UK. The disease was contracted either in an indoor swimming pool, geothermal bath, or in a stream thermally polluted by the effluents of an industrial plant. For instance, 16 patients in the Czech Republic and four in Belgium contracted the disease in an indoor swimming pool; however, investigators could not find traces of //N. fowleri// from the swimming pools involved in Belgium. In addition, one patient in Italy contracted the disease after swimming in a river and three others in the UK after swimming in a geothermal bath. While no other countries in Europe have reported cases of PAM, //N. fowleri// has been found repeatedly throughout the rest of Europe, mainly in France ** [4] **<span style="font-family: Arial,Helvetica,sans-serif;">.

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;"> **Cases in North and South America** <span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;"> There have been 120 confirmed cases of PAM in North America: 111 cases have been reported in the USA, and nine in Mexico. In the USA, //N. fowleri// infection predominately occurs in the southern states. Most patients contracted the disease after swimming in warm freshwater bodies. In South America, PAM was reported only in Venezuela and Brazil, seven and five cases respectively. In Venezuela, 7 patients contracted the disease<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12.5px;"> after swimming in natural warm lakes or untreated pools. In Brazil, five cases were linked to an artificial lake ** [1,4,5] **<span style="font-family: Arial,Helvetica,sans-serif;">.