Naegleria fowleri, a free-living ameba, is the causal agent of primary amebic meningoencephalitis (PAM), which is an acute, fulminant, and rapidly fatal infection of the central nervous system (CNS). N fowleri is named after Malcolm Fowler, an Australian pathologist, who first isolated it from a patient with PAM.[1] PAM develops following several days of exposure to the contaminated water source and typically causes death within 1-2 weeks after admittance to the hospital. Few individuals survive the infection, partly because of its rapid onset and partly because of delayed diagnosis. Only around 10 survivors of N fowleri PAM have been reported in literature.[2]
Bull coined the term “primary amebic meningoencephalitis” to distinguish it from the secondary meningoencephalitis caused by the intestinal ameba Entamoeba histolytica.
Naegleria species are amebo-flagellates that are ubiquitous in soil and fresh or brackish water (lakes, rivers, ponds). In general, they are sensitive to environmental conditions such as aridity and pH extremes and cannot survive in seawater. In humans, they are found in the throat and nasal cavity. N fowleri is heat-tolerant and is able to survive temperatures up to 45.8°C, pre-adapting the species to mammalian body temperature. Hence, an incubation temperature of 45°C is routinely used to isolate N fowleri from water samples, while suppressing growth of other amebae in the samples.
Although 30 species of Naegleria have been recognized based on sequencing data, N fowleri is the only one that has been isolated in cases of amebic meningoencephalitis. Other Naegleria species (Naegleria australiensis, Naegleria italica, Naegleria philippinensis) have been found to be pathogenic in murine models of PAM but have not been identified in any human cases of the infection.[3] As it grows best at elevated temperatures, N fowleri has been isolated from warm-water bodies, including man-made lakes and ponds, hot springs, and thermally polluted streams and rivers. It feeds on bacteria in the water bodies for survival. N fowleri is not found in saltwater sources such as seawater.[1] In the United States, most N fowleri infections occur in the summer months.[4]
Life cycle of N fowleri
The life cycle of N fowleri consists of 3 stages: trophozoite, a temporary flagellar stage known as amebo-flagellate, and cyst.
The trophozoite is the vegetative or feeding stage of the ameba and is the infective form. In humans, this form is found in CSF or in tissue. It measures 10-20 µm in diameter and has a granular cytoplasm and a distinct ectoplasm. The trophozoite is characterized by a large central nuclear karyosome surrounded by a halo. Trophozoites are actively motile with the help of a broadly rounded, granule-free projection called lobopodium that originates from the surface. The projection helps to ingest bacteria, yeast cells, and cellular debris and may serve as an organelle of attachment. In tissue, trophozoites ingest red and white blood cells and cause tissue destruction, and this stage is the only one in which the ameba multiplies via binary fission.
The flagellate stage, also known as the amebo-flagellate stage, is a temporary form of the ameba in which it neither feeds nor divides in culture. The ameba progresses to the amebo-flagellate stage when the trophozoites form is exposed to a change in ionic concentration, such as in distilled water. During the amebo-flagellate stage, the parasite is pear-shaped with a flagellar apparatus at the broader end. The flagellar apparatus consists of 2 terminal flagella, 2 basal bodies, microtubules, and a single striated rootlet, or rhizoplast. During the flagellated stage, the parasite may exhibit a rapid forward movement or a slowly spinning circular movement. It reverts to the trophozoite stage within 24 hours.
The cystic stage represents the resistant form of the parasite, offering protection from desiccation and food shortage. The cyst is round, measures 7-10 µm in diameter, and is surrounded by a smooth double-layered 1-µm wall. The cyst consists of a single nucleus, contractile vacuoles, and food vacuoles. In stained preparations, only the vacuoles can be demonstrated as fine granules, but not the nucleus. Cysts are usually absent in clinical specimens, as the infection is so rapid and fatal that the patient typically dies before the trophozoites encyst.[5]
N fowleri infections in humans occur while swimming or diving in warm water contaminated with the parasite. The parasite invades its host by penetrating the olfactory mucosa. Human-to-human transmission has never been reported. During the initial stages of infection, the host response is initiated by the secretion of mucus that traps the trophozoites. Despite this response, some trophozoites are able to reach, adhere to, and penetrate the epithelium.
It secretes a 37-kda protein with mucinolytic activity, which degrades the mucus on the surface of the nasal mucosa, thereby facilitating the invasion of the organism.[6] The gene nfa1 has been isolated from the free-living pathogenic amoeba. The protein Nfa1, which has cytolytic function, is located in pseudopodia and specifically in food-cups. N fowleri trophozoites are neurotrophic.[7] They enter the nose and invade the olfactory mucosa and bulbs, penetrate the submucosal nervous plexus, invade the cribriform plate, and reach the subarachnoid space. Glucose and protein in the CSF support the growth and multiplication of the amebae. The high content of oxygen in the CSF and in the brain also facilitates growth of the amebae.
The trophozoites enter the ventricular system through the foramen of Luschka and Magendie and reach the choroid plexus. These then destroy the ependymal layer of the third, fourth, and lateral ventricles and produce acute ependymitis. They multiply by a process known as promitosis, during which an intact nuclear membrane (demonstrable on electron microscopy) is present. Only trophozoites are found in pathologic lesions in humans.
Most patients with PAM have a history of swimming or diving in a body of fresh water. In arid climates, some cases of PAM have been attributable to the inhalation of cysts. Trophozoites or cysts, which give rise to trophozoites after they excyst, penetrate the nasal mucosa and ascend along the olfactory nerves after phagocytosis by sustentacular cells of the neuroepithelium. Subsequently, they pass through the cribriform plate to invade brain tissue, with resultant purulent meningitis and encephalitis.
The course of N fowleri infection is fulminant and rapid, and patients with PAM die usually within a short period (5-10 d). Therefore, detectable levels of specific antibodies are not produced in the serum during the disease. The role of the cell-mediated immunity (CMI) in resistance to N fowleri infection is not fully understood.
United States
Although isolation of N fowleri from the nares of asymptomatic individuals has been reported, PAM itself is rare in the United States. From 1962 to 2016, 143 cases have been reported from the United States.[1] Almost all of these have occurred in the Southern tier states, with the majority from Texas and Florida.[8] Of late, few cases have been reported from states beyond the Southern tier such as Minnesota, Kansas, and Virginia. Hence, a possibility of a change in trend in the epidemiology of PAM in the United States is suggested.[8] On an average, 0-8 human infections occur in the United States every year.[4]
Although most N fowleri infections are contracted by swimming in infested fresh water collections, recent reports suggest that the use of ”neti pots” for nasal irrigation can also predispose to PAM. In 2011, 2 adults died in Louisiana due to PAM. Both the patients did not have any history of swimming in the recent weeks. Their only reported exposure to water was the regular use of neti pots for sinus irrigation. N fowleri was identified in the tap water samples taken from the homes of both these patients.[9]
In a survey of 16 sites from Lake Anna in Virginia, a lake that is used to cool reactors at a nuclear power plant and for recreational activities, sampled during the summer of 2007, 9 were found to be positive for N fowleri by a nested polymerase chain reaction (PCR) assay. However, total ameba counts, inclusive of N fowleri, never exceeded 12/50 mL of lake water at any site. The presence of N fowleri was not found to correlate with the conductivity, dissolved oxygen, temperature, or pH of water. To date, no cases of PAM have been reported from this thermally enriched lake.[10]
Naegleria species have been in detected in domestic water supplies in the north-central United States[11] and in warm ground water aquifers.[12] However, PAM cannot be acquired by drinking water contaminated with Naegleria. Infection can occur only when the contaminated water enters the nose.[1] Over 10 years, from 2007-2016, a total of 40 cases were reported in the United States, including one case in 2016 involving a 16-year-old boy.[1]
International
The risk of infection has been estimated at 1 case per 2.6 million exposures to N fowleri. Until 2008, at least 440 cases of PAM due to N fowleri were reported worldwide, with some well-publicized outbreaks related to a single source.[13] Most cases occur during the warm summer months in individuals who swim in freshwater pools or lakes.
Most worldwide cases have been reported in the United States. Other cases of N fowleri infection have been reported in the Czech Republic, Australia, Mexico, New Zealand, Nigeria, Great Britain, and India. Over a 20 cases have been reported so far from different parts of India.[5, 13, 14] Most recently, a 25-day-old neonate, the youngest survivor of PAM, has been reported from India.[2]
In 2011, Shakoor et al reported 13 PAM cases from Pakistan; the patients had no history of aquatic activities and the proposed mode of infection was ablution with tap water[15] In 2015, 12 fatal PAM cases were reported in the Sindh province.[16]
A case of PAM has been reported from Vietnam, where the patient had acquired the infection due to freshwater pearl diving.[17]
Naegleria species have also been isolated from swimming pools in Malaysia,[18] from recreational waters in Taiwan,[19] and from natural water sources supplying the cities in Turkey.[20] In South Korea, Naegleria isolates have been reported from sewage, water bottles, freshwater fish, and clinical isolates such as corneal scrapings.[21] The first case of PAM in Zambia was reported in 2017.[22]
Mortality/Morbidity
PAM carries a mortality rate of greater than 95%, with death occurring within 4-6 days. Of all the 143 cases of PAM reported in the United States from 1962-2016, only 4 patients have survived, one in 1978, two in 2013, and one in 2016.[1] Very few survivors of PAM have been reported from other countries, but include Mexico and India. Only 8 survivors have been reported worldwide until 2005. Three more survivors have been reported from India in 2008, 2012, and 2013.[2, 23, 24, 25] In the rare cases of survival, PAM was recognized very early, allowing for early institution of aggressive therapy. In one person who survived, the N fowleri strain was found to be less virulent than the strains recovered from other fatal cases.[26] Most survivors have some residual physical or cognitive impairment. One recent survivor was an 8-month-old male infant from India who was treated vigorously and avoided any neurological deficit.[24]
PAM has no racial predilection.
PAM has a male-to-female ratio of 3:1. Although PAM has no natural sexual predilection, the predominance in males is thought to be secondary to a greater risk of exposure due to behavioral factors.
Most cases of PAM have been reported in children and young adults, presumably because of a greater exposure risk in these populations due to behavioral factors. In addition, the more porous cribriform plate in children and young adults is thought to place these individuals at a higher risk for disease.
Most cases of PAM involve a history of exposure to fresh, warm water. Patients with PAM generally present with a history of bathing in a pond or lake 2-6 days prior to the onset of symptoms of meningeal irritation.
The index of suspicion should be increased in children and young adults who have a history of recently swimming in freshwater lakes, ponds, and pools. Isolated cases of PAM have followed bathing in tap and hot water.
Clusters of cases of PAM have been documented during the summer months when freshwater sources are warm.[27] Although numerous people are exposed to the amoeba while swimming, it is unclear why that certain people are affected and most are not. Attempts have been made to find out the concentration of free-living N fowleri in recreational lakes and to determine the standard cut-off concentration for risk of contracting infection.[28] However, no such standard is available to date.
Most cases of primary amebic meningoencephalitis (PAM) involve a history of exposure to fresh warm water. Patients with PAM generally present with a history of bathing in a pond or lake 2-6 days prior to the onset of symptoms of meningeal irritation.
The index of suspicion should be increased in children and young adults who have a history of recently swimming in freshwater lakes, ponds, and pools. Isolated cases of PAM have followed bathing in tap and hot water.
Clusters of cases of PAM have been documented during the summer months when freshwater sources are warm. The higher temperature during the hot summer months facilitates the growth of N fowleri.
The physical signs of PAM are similar to those of bacterial meningitis, as follows:
PAM is caused by infection with the ameba N fowleri. In 2009, another free-living amoebae, Paravahlkampfia francinae, has been reported to cause PAM.[29]
PAM is typically fatal. Death is due to pulmonary edema or cardiorespiratory arrest within a week of appearance of the first symptoms. Persistent seizures may occur in patients who have otherwise recovered.
The diagnosis of primary amebic meningoencephalitis (PAM) is always parasitic and is based on detection and identification of N fowleri trophozoites in the CSF or brain biopsy samples.
The CSF is the specimen of choice for demonstration of the amebae. Brain tissue biopsy can also be used for detection of antigen.[30]
The CSF is centrifuged at 150 xg for 5 minutes. The supernatant is aspirated, and the sediment is suspended in the remaining fluid. A drop of sediment suspension is kept on a slide and mounted with a coverslip and is examined with compound light microscopy using 10X and 40X objectives. The specimen is best examined with phase contrast microscopy. This may show trophozoites with lobopodia extension and retraction.
The amebae are detected based on their active directional movements. Close observation is important because PAM can be diagnosed based on the observation of trophozoites; however, these have been confused with WBCs in reported cases. Cyst and flagellated stages are not found in CSF samples; if both cysts and trophozoites are found in CSF, it is highly suggestive of Acanthamoeba infection, ruling out Naegleria PAM.
A few drops of CSF are mixed with 1 mL of distilled water and examined after 1 hour for flagellated forms typical of N fowleri. Trophozoites of N fowleri measure around 10-25 μm, with typical limacine/eruptive amoeboid movement, indicating a positive enflagellation test result.[31]
CSF Gram stain findings are usually negative. RBCs are present. CSF stained with Wright-Giemsa or modified trichrome stain may show trophozoites with large karyosome and a contractile vacuole.[1] Direct fluorescent antibody staining of CSF smears is useful for demonstrating N fowleri in the CSF.
Naegleria species can be readily cultivated on either nonnutrient agar or agar media containing low concentrations of nutrients (eg, peptone 0.05%, yeast extract 0.05%, glucose 0.1%) in the presence of living or killed bacteria or in defined axenic media, as proposed by Chang et al and Nerad et al, among others. The culture plate can be incubated at 42°C, which facilitates the growth of only the thermophilic amoebae while killing the other free-living amoebae.[1] A nonnutrient/low-nutrient agar is chosen to prevent overgrowth of bacteria.[32] In general, the bacteria of choice include nonmucoid strains of Klebsiella pneumoniae, Enterobacter species (Enterobacter aerogenes and Enterobacter cloacae), and Escherichia coli. After several days, the plate is microscopically inspected; Naegleria cysts are identified by trails left by migrating amebae in the lawn of the bacteria. Various molecular methods can be used for final confirmation of the identity of the species.
In 2017, a liquid encystment medium modified from Page’s amoeba saline was reported to be useful for obtaining pure N fowleri cysts.[33]
Serologic testing has no role in the diagnosis of acute PAM, since little time is available from onset to death to mount an antibody response. In one survivor, detectable antibody persisted for more than 4 years.
PCR is available at some research sites using numerous primers. Molecular characterization of strains is also useful in tracking infections to a source and in recognizing potential risks for swimmers or bathers in particular locales. A species-specific DNA probe is available to identify N fowleri in environmental samples, followed by restriction fragment length polymorphism (RFLP) analyses of whole-cell DNA for confirmation. Epidemiologic typing of N fowleri was used in an analysis of the 5.8S rRNA gene and the internal transcribed spacer (ITS) of clinical isolates. In a study performed in the United States, a rapid, sensitive, and specific assay for the detection of N fowleri was developed using Mp2C15 probe in a nested PCR assay format.[34] A nested PCR assay has also been applied to detect the presence of the parasite in domestic water sources.[35]
Recently, flow cytometry has been used for the diagnosis of N fowleri infection.[36] Flores et al evaluated flow cytometry and monoclonal antibodies in differentiating Naegleria fowleri from Acanthamoeba species.[37]
Lately, a real-time PCR using hybridization fluorescent-labelled probes, targeting the N fowleri Mp2Cl5 gene sequence, has been developed. The reaction detection limit in their study was 1 copy of the Mp2Cl5 DNA sequence.[38]
Visvesvara has reported the development of a multiplex real-time PCR that could simultaneously look for Naegleria, Acanthamoeba, and Balamuthia species in a single specimen, thus reducing the time for diagnosis. This is especially useful as infection with any of the 3 amoebae could have clinical presentations indistinguishable from each other.[39] Similarly, Qvarnstrom et al described a TaqMan-based multiplex real-time PCR that targets the 18S rRNA gene in the detection of N fowleri, Acanthamoeba species, and Balamuthia mandrillaris.[40]
Both immunofluorescence and immunoperoxidase methods are useful for demonstrating N fowleri trophozoites in the histologic sections of the brain biopsy samples.[30]
Head CT scanning yields nonspecific findings, showing a loss of the subarachnoid space and diffuse gray material enhancement.
CSF studies show the following:
Lumbar puncture: Wet-mount examination of CSF is the main diagnostic tool in PAM.
N fowleri infection produces lesions mainly in the base of the brain, brain stem, and cerebellum. The olfactory mucosa and bulbs are the most commonly affected areas. The lesions consist of an acute necrotizing meningoencephalitis associated with moderately purulent exudates. Only trophozoites are found in the CNS lesions, not cysts
Early diagnosis, treatment, and aggressive supportive care hold the only chance for survival in patients with primary amebic meningoencephalitis (PAM). Very few survivors have been reported, with probably no more than a dozen survivors of an estimated 200 cases.
The best described and authenticated case of successful treatment of PAM involved a 9-year-old girl who was diagnosed early in the disease course and was treated with intravenous and intrathecal amphotericin B, intravenous and intrathecal miconazole, and oral rifampin. The patient survived with minimal neurologic sequelae.[23] In addition, the most recent survivor from India, a 25-day-old boy, was successfully treated with the regimen comprising of amphotericin B, rifampicin, and fluconazole for 4 weeks, along with ventriculoperitoneal shunt for obstructive hydrocephalous. The child was free of neurological sequelae at 8-month of follow-up.[2]
The variables in determining the survival likelihood include how early the diagnosis is made and treatment initiated, the infectious dose of amebae, the virulence of the infecting strain, and the health of the patient.
Naegleria species are highly sensitive to the antifungal drug amphotericin B, and it has been used as the core antimicrobial in virtually all cases in which recovery occurred. In a 2017 study, amphotericin B was shown to induce apoptosislike programmed cell death in Naegleria.[41] Minimum amebacidal concentrations of amphotericin B were determined to be 0.02–0.078 µg/mL. Ultrastructural examination of amebae treated with amphotericin B revealed membrane distortions, including the nuclear envelope, rough and smooth endoplasmic reticula, and plasma membrane blebbing.
The macrolide antibiotic azithromycin is effective against Naegleria species in vitro and in murine models, but it has been reported to have poor CSF penetrance. Other antimicrobials that have been tested, mostly in vitro, include clotrimazole, itraconazole, fluconazole, and ketoconazole, with varying degrees of efficacy. Differences in reported drug sensitivities are due to the use of different N fowleri strains in different laboratories, which show variation to drugs. However, amphotericin B remains the drug of choice in the treatment of PAM.
Other drugs that have been evaluated for the treatment of meningoencephalitis due to Naegleria species are miltefosine and chlorpromazine. Kim et al studied the effect of these 2 agents on N fowleri both in vitro as well as in vivo in mice models and compared them with the therapeutic effect of amphotericin B. Chlorpromazine was found to have the best therapeutic activity against Naegleria, both in vitro and in vivo.[42]
In conjunction with the FDA, the CDC has an expanded access investigational new drug (IND) protocol in effect to make miltefosine available directly from the CDC for treatment of free-living amebae (FLA) in the United States. These infections include primary amebic meningoencephalitis (PAM) caused by N fowleri and granulomatous amebic encephalitis caused by B mandrillaris and Acanthamoeba species.[43] All three recent survivors of PAM in the United States (two in 2013 and one in 2016) were treated with miltefosine.[1] Various other agents have been studied in vitro and/or in vivo, including hygromycin, rokitamycin, clarithromycin, erythromycin, roxithromycin, and Zeocin.[44]
Owing to the increasing number of cases being reported and the controversial efficacy of amphotericin B, active research is being carried out to identify newer drugs that are more effective with fewer adverse effects. Recently by using high throughput screening, Debnath et al have identified a novel macrolide, corifungin, with higher activity against Naegleria than amphotericin B. By using in vitro and in vivo animal studies, the authors have demonstrated the superiority of corifungin over amphotericin B. Based on these results, corifungin has been given an orphan drug status by the US Food and Drug Administration for the treatment of PAM.[45]
Upon evidence of increased intracranial pressure and possible herniation, emergent consultation with a neurosurgeon is warranted for ventriculostomy.
Consult with an infectious disease specialist early in the course of illness.
The only definite way to evade N fowleri infections is to abstain from water-related activities.[4]
Since N fowleri trophozoites and cysts are susceptible to chlorine, swimming pools should be adequately chlorinated. Case clusters have been reported in association with poorly chlorinated pools. Strongly consider closure of a site if a case of PAM occurs. Warm water is known to be more at risk of harboring these organisms. Thus, avoiding activities in bodies of warm fresh water, hot springs, and water around power plants, which may be thermally polluted, may be advisable. In addition, when taking part in such activities, it would be better to avoid the accidental entry of water into the nose. There are no public health criteria established for what levels of amebae present are acceptable or unacceptable.[46]
People with the habit of using neti pots should use distilled, boiled, or filtered water. The neti pots must be thoroughly washed before and after use.[9]
No vaccine is currently available for the prevention of PAM due to N fowleri. However, owing to the rise in the number of infections, vaccine research is being carried out. Through animal experiments using mice, the efficacy 2 vaccine candidate proteins have been recognized. Intranasal administration of Cry1Ac protoxin along with amoebic lysates has been found to elicit a protective Th2 response against N fowleri infection.[47] Intraperitoneal administration of recombinant Nfa1 protein has been found to prolong the mean survival time of mice when challenged with infection.[48]
Patients with primary amebic meningoencephalitis (PAM) should be monitored in the ICU.
The goals of pharmacotherapy are to eradicate the infection, to reduce morbidity, and to prevent complications.
Amphotericin B offers the only clear evidence for a survival benefit in the treatment of primary amebic meningoencephalitis (PAM).
Clinical Context: Amphotericin B is a polyene antibiotic produced by a strain of Streptomyces nodosus; it can be fungistatic or fungicidal. Amphotericin B binds to sterols, such as ergosterol, in the fungal cell membrane, causing intracellular components to leak, with subsequent fungal cell death.
The mechanism of action usually involves inhibiting pathways (enzymes, substrates, transport) necessary for sterol/cell membrane synthesis or altering the permeability of the cell membrane (polyenes) of the fungal cell.
Hematoxin and eosin (H&E)-stained photomicrograph (125X) that shows the cytoarchitectural histopathology found in a case of primary amoebic meningoencephalitis (PAM), caused by Naegleria gruberi. Courtesy of the US Centers for Disease Control and Prevention and Dr. George R. Healy.
