N. Fowleri Diagnosis, Treatment and Prevention

Historical Aspect:

Naegleria Fowleri (N. Fowleri ) is a free living, thermophilic protozoan that is a human specific pathogen that attacks the central nervous system. It can be found in contaminated freshwater sources. It enters through the nose and travels to the brain causing primary amoebic meningoencephalitis¹. It was first observed in 1899 and later named after Dr. M. Fowler, who observed the first reported fatal cases of acute pyogenic meningitis in Australia in 1965². While these infections have been identified as early as the 19^th century it is challenging to identify because it mimics many of the symptoms of bacterial meningitis[1].

Primary amoebic meningoencephalitis (PAM) is a necrotizing and hemorrhagic meningoencephalitis³. The symptoms begin 1-9 days after the onset of infection these symptoms include fever, nausea, headache and vomiting. The initial symptoms mimic those of bacterial meningitis, the later symptoms are unique to this disease. Later symptoms include neck stiffness, hallucinations, seizures, an inability to focus, lack of balance and eventually coma and death. The mortality rate for this disease is 95%. The disease progresses quickly and leads to death within 12 days of the initial infection¹.

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While this infection has a high mortality rate, it is very rare. There have been 300 reported cases of PAM worldwide in the last 40 years⁴. It is important to note that this conditioned is often misdiagnosed so these numbers are estimates. In the United States there have been 138 cases in the last 50 years¹. This infection was once a condition that plagued developing countries but the incidence is spreading all over the world. Researchers suspect that the increased temperatures due to global warming, increased use of public water sources due to scarcity and an overall increase in aquatic recreational activities are to blame^3,18.

While swimming and other aquatic recreational activities help proliferate this disease so do rituals. Aga Khan University in Pakistan noticed an increased number of deaths caused by PAM in young males that had no history of swimming, but were devout Muslims. Those who practice this faith pray five times a day and before every prayer, they perform ablution, ablution is the washing of the hands, face, ears, nose, mouth, arms and feet. While cleansing the nose, water is forced up the nose putting individuals performing this practice with inadequately purified water at a greater risk for PAM³. Religious festivals like the Kumbh Mela where Hindus gather and swim in the Ganges river put those who participate at risk of developing diseases like PAM caused by N. Fowleri ³. In addition to religious practices, therapeutic interventions like the Neti pot increase the risk of PAM. Nasal irrigation systems like the Neti pot work to relieve the symptoms of sinusitis and cold. It works by removing debris and mucus from the nasal passages. The recommendation is that the water should be boiled or mixed with a non-ionized sodium chloride.

Basic structure:

N. Fowleri are a part of the free living amoeba that cause infections in the central nervous system. Some of the other protists are Acanthamoeba spp and Balamuthia mandrillas. Naegleria fowleri have been classified by modern techniques which analyze morphology, biochemical pathway and molecular phylogeny². The modern approach classifies N. Fowleri as a part of the super group Excavata, in the group Heterolobosea and a part of the family Vahlkampfiidae. Although the genome for N. Fowleri is not yet completed there are some studies producing information about its molecular and genetic characteristics. N. Fowleri 's genus includes more than 40 species, but N. Fowleri is the only one that is known to cause disease in humans. De Jonckheere created the most popular identification system for N. Fowleri . The identification system uses genetic markers like internal transcribed spacers (ITS1) and 5.8S rDNA ². This identification system revealed at least 8 different genotypes. The genotypes are dispersed among different continents America (I,II,III), Europe (III,IV,V,VI,VIII), Oceania (V), and Asia (II,III). Of the eight genotypes only four have been found in humans, types 1-4².

Naegleria are a part of the group heteroloboseans that have a three-phased lifecycle. They are first amoeba, then flagellate and lastly cyst formation⁵. N. Fowleri reproduces in the amoeba form via binary fission to produce the cyst and the flagellate forms. The entire cell cycle is 8 hours, N. Fowleri spends 28 minutes in M phase, 180 minutes in G1, 183 minutes in S phase, and 90 minutes in G2². In the amoeba form, the trophozoite ranges in size from 15-25 µm. Trophozoites also have cytoplasmic projections called food cups which allow phagocytosis of bacteria, yeast, erythrocytes and cellular waste. Trophozoites are the form of the amoeba that can feed and divide, they are also the form that enter the human host⁶.Trophozoites will transition into the flagellate stage after being exposed to a saline solution². The flagellates cannot feed or divide, the transition also involves a change in shape from pleomorphic to pear shaped with a pair of flagella. The flagella have the typical 9+2 structure and are surrounded by a cytoplasmic membrane. The 9+2 flagella structure describes the cross- sectional arrangement of microtubules that make up the flagella. There are nine doublet outer tubules and two central singlet tubules⁷. The cyst form is resistant to most disinfection. The cyst formation is spherical, smooth, double walled and refractive. They measure about 20µm. The material of the cyst wall is synthesized and packaged by the rough endoplasmic reticulum².

Route of Transmission:

N. Fowleri is a thermophilic amoeba, its optimal temperature ranges from 115⁰ to 122⁰ F. N. Fowleri can typically be found in warm freshwater like lakes and rivers, warm water from industrial parks, or inadequately chemically treated water, other warm water sources like water heaters and soil. In their natural environment N.Fowleri phagocytize cyanobacteria and eubacteria to regulate levels.

Samples from the lakes of the southern United States reveal that N. Fowleri introphozoite formis present during the summer. During the winter months N. Fowleri in cyst form survive in freshwater sources, but no form of N. Fowleri can withstand freezing temperatures¹ .

Most cases of PAM are caused by swimming in warm freshwater, from drinking water, recreational activities, ritual abulation and sinus irrigation systems¹. Infection occurs when water containing N. Fowleri gets into the nose. The amoeba enters the nose and travels along the olfactory nerve, through a bony plate in the skull called the cribriform plate³. Once it reaches the brain it causes meningoencephalitis, cerebral edema and results in herniation. The olfactory bulbs and orbitofrontal cortices become necrotic and hemorrhagic. The data on both humans and mice support the conclusion that death is ultimately caused by increased intracranial pressure and herniation³.

Swimming in water containing N. Fowleri increases the risk for PAM but age and sex are also risk factors. From the 1962- 2015 there have been 138 reported cases of PAM in the United States, 114 of the cases have been children around the age of 12. Nearly 75% of the infections have affected males¹. Certain behaviors are associated with an increased risk of infection, those infected individuals reported participating in water related activities like swimming, diving and head dunking¹. Although N. Fowleri can be transmitted through water it cannot be transmitted through aerosols or droplets, or via person to person contact. N. Fowleri can be found in other organs of the body, such as the heart, lung, spleen and thyroid¹.

Pathogenesis:

N. Fowleri enter the human host through the nose which provides access to the brain. Within eight hours of infection N. Fowleri is present in the mucus layer of the olfactory epithelium. Within 24 hours N. Fowleri are in the olfactory bulb and present in the cribriform plate. By 96 hours neutrophil polymorphs cause a severe inflammatory response in the olfactory bulb which leads to brain tissue damage³.

Contact dependent mechanisms are N. Fowleri mediated pathogenic processes. The primary mechanism of pathogenesis in N. Fowleri is adhesion. Adhesion allows for movement and chemotaxis in the nasal mucosa and assists N. Fowleri with disease progression. Adhesins are expressed on the surface of N. Fowleri, the adhesins are integrin like proteins surrounded by adhesion like structures. Fibronectin binding protein, protein kinase C and NFa1 are important to interrupting the host mediated cytotoxicity^3. In an experiment testing cytopathicity of N. Fowleri, a culture would bind to Fibronectin and in the presence protein kinase C the ability of the amoeba to adhere increased⁸. N. Fowleri also produces Reactive Oxygen Species (ROS) which damage the host cell. Following cell damage, N. Fowleri uses phagocytosis and amoebastomes to assist N. Fowleri in breaking down and consuming the cells through a sucker structure on its surface. These processes are mediated via actin and involve the polymerization of monomeric G-actin and filamentous F-actin. Studies have found that a membrane protein Mp2CL5 may also play a role in pathogenicity, without this protein N.Fowleri are nonpathogenic³ .This protein is suspected to aid in pathogenicity by navigating the environment, and movement toward food sources⁹.

In addition to contact dependent mechanisms of pathogenicity, N.Fowleri also utilizes contact independent mechanisms. N-PFP is a cytolytic pore forming protein that depolarizes the cell membrane and decreases the integrity. Naegleriapores A and B are pore forming polypeptides that are very similar in structure and function. Both are antimicrobial and cytolytic polypeptides³. The enzymes phospholipase A, A ₂ and C are present in patients with PAM. Phospholipases are responsible for the demyelination of white matter. Sphingomyelinase, neuroaminidase, elastase and proteolytic enzymes are responsible for demyelinating nerve tissue. N. Fowleri are hemolytic due to the heat shock protein 70 which is unaffected by salt concentrations, chelating agents, pH and temperature extremes^3,10. This protein is present in the cytoplasm, pseudopodia and phagocytic food cups. There are many other factors associated with the pathogenicity of N. Fowleri and others that are suspected to have an effect on the pathogenicity.

On the onset of infection the host's innate immune system attempts to reduce the pathogen's cytotoxicity. During the early infection the body releases mucin which surrounds the N.Fowleri trophozoites to prevent cytotoxicity. In the later infection eosinophils and neutrophils surround the N. Fowleri cells to prevent cytotoxicity. Inflammation increases over time, although there are not many cells that penetrate the host epithelium. The inflammation and polymorph nuclear cells from the host response damage cerebral tissue² .

Diagnosis, Treatment, Prevention:

Diagnosis of N. Fowleri is heavily dependent on laboratory techniques. The most effective way to diagnose N. Fowleri requires cerebrospinal fluid (csf) which is conducted while the patient is living and brain biopsy which is conducted post-mortem¹¹. Different laboratory tests are utilized to analyze the specimen. When PAM is suspected, samples can be wet mounted and placed under a microscope to identify trophozoites¹². Polymerase chain reaction is a method that can be used to amplify DNA, to identify the presence of N. Fowleri DNA in a sample¹¹. Another laboratory technique involves antigens that were developed from mouse monoclonal antibodies (mAbs) against N. Fowleri . When indirect immunofluorescence assays are used mAbs react to N.Fowleri from specific geographic regions¹³.

The infection due to PAM progresses quickly and as previously stated, mimics symptoms of bacterial meningitis. Even with the advances in laboratory diagnostics most cases are diagnosed post mortem making effective treatment elusive. One successful case study provides an example of effective diagnosis and treatment of this condition. On July 13, 2013, a 12 year-old girl presented to Arkansas Children's Hospital vomiting, having trouble holding up her head and was unable to open her eyes. A few days prior to hospitalization the patient had been playing in a local water park. During her hospitalization she experienced hallucinations, and thirst. A spinal tap was performed which ruled out bacterial meningitis. The laboratory identified N. Fowleri trophozoites in the patient's cerebrospinal fluid. After determining the infection was PAM caused by N. Fowleri, physicians initially treated the patient with antibiotics and antifungals like Amphotericin B, Rifampin, Fluconazole, Dexamethasone and Azithromycin .None of these treatments improved the condition of the patient. The hospital petitioned the Center for Disease Control (CDC) to allow the use of a new experimental drug available for the treatment of N.Fowleri¹⁴. The drug Miltesfosine was given 36 hours after the initial diagnosis, physicians also lowered the patient's body temperature to 93.2 F⁰ to reduce cerebral edema and intracranial pressure. After 18 days in the ICU there was no N. Fowleri found in her system. The patient experienced a full but gradual recovery over the next fifty five days. After seven days the patient was able to write her name, in fourteen days she was able to speak in one and two syllable words. She also underwent both speech and physical therapy¹⁴.

This patient is one of the three known survivors of PAM. While the virulence factors and the degree of recovery that surround the other two cases of survival are unknown. The prompt diagnosis, treatment with Miltesfosine within thirty six hours and maintaining a low body temperature for this patient played significant roles in effectively treating this infection¹⁴. Although the first case of N. Fowleri was over 50 years ago, the mortality rate for this disease continues to increase due to water scarcity which increases use of water from public sources. As previously stated this condition is either diagnosed post mortem or misdiagnosed. The development of a standard microbial treatment will aid in the reduction of high mortality rates¹⁴.

In the three cases of survival the patients were all intially treated with amphotericin B, rifampcin, fluconazole, dexamethasone and phenytoin during the first week of infection¹⁵. In 1969 a patient survived PAM with the successful treatment of amphotericin B. The patient in 2013 was initially treated with amphotericin B and it was ineffective. Miltesfosine effectively treated this patient's PAM¹⁴. Other drugs with the potential to treat PAM have been tested, and some have been proven effective while others have not. Clotrimazole a drug that has been used as an antifungal had potential to treat PAM but under further study was deemed ineffective¹⁶.

In developing countries like Pakistan where water is in short supply and ablution is common practice the danger of becoming infected with N. Fowleri is greatly increased. Water sources in these countries include wells or water storage tanks which are often contaminated with N. Fowleri ¹⁷. In order to prevent infection the World Health Organization (WHO) encourages that water storage units and wells be regularly tested to ensure proper disinfection. Public health organizations have also encouraged the use of nose clips while swimming in lakes and other freshwater sources, and boiling water that is used for ablution¹⁷.

Chlorine disinfection regimens prevent against most pathogens in drinking water systems however free living amoeba like N. Fowleri survive most disinfection. The cyst form of N. Fowleri is resistant to most disinfection and are associated with biofilm that can build up in drinking water systems. N. Fowleri have been isolated in drinking water systems in Australia, the United States and Pakistan, in both Australia and the United States they maintain chlorine levels of 0.5mg/L at all times in the drinking water¹⁸.To test the amount of chlorine needed to eliminate N. Fowleri, researchers conducted an experiment using two separate sites, a pre re-chlorination site and a post re-chlorination site, both sites were monitored before and after re-chlorination for a year. The results were that after chlorination of greater than 1mg/L at each site, in the post re-chlorination site the amoeba were gone within 60 days. The pre re-chlorination site would have seasonal flares of N.Fowleri but the chlorine levels eliminated the protozoan and prevented further spread. Overall chlorine levels above 1mg/L result in the elimination of N.Fowleri in drinking water systems¹⁸.

Summary of current areas of research notes:

N. Fowleri is a rare pathogen that was discovered over fifty years ago. Since its discovery still not much is known about this pathogen. Future research into this pathogen will focus on patient complaint diagnosis and treatment, expanding the drugs that are used, biomarkers, and drug targets. In order to determine whether the patient has contracted PAM due to N.Fowleri the patients csf is tested and if the test is negative for bacterial cultures and the patient has a history of swimming or other aquatic activities, then the patient tests positive for N.Fowleri. Extracting csf can increase the pressure in the patient's brain and lead to herniation of the brain. Because N. Fowleri travels to the brain via the nasal passage, the proposed route of diagnosis is collecting a nasal sample. Research confirms that N.Fowleri can be collected in both csf and nasal cultures³.

Drugs administered through the nasal cavity, through the transcribial route would be delivered across the cribifrom plate to the inferior portion of the frontal lobe. This is the site where N. Fowleri attacks and spreads to the central nervous system. Drugs like amphotericin B do not decrease the minimum inhibitory concentration (mic) when administered intravenously. By potentially administering the drug transcribialy, the drug passes the blood barrier which would "allow the drug to be more potent, trail the route of N.Fowleri, attack the site of infection, allow the lethal dose of drug to achieve the mic without venous drainage, and lastly to avoid symptoms of intravenous drug administration" ¹⁵.

There are clinically approved drugs that have promising amoebicidal effects. These drugs interrupt the mechanisms and processes of the amoeba. Digoxin and proyclidine both exhibit amoebicidal properties. Digoxin treats atrial fibrillation and heart rhythm disorder by helping the heart beat stronger and with more rhythm¹⁹. Proyclidine is used to treat Parkinson's and other diseases that cause involuntary muscle movement²⁰. In order for further testing of the amoebicidal effects of these drugs to continue to be studied more drugs that have the potential to be amoebicidal must be identified and screened for tests to go from in vitro testing to in vivo testing. There has not been a lot of emphasis on finding drugs that treat N. Fowleri because the condition is rare and affects populations in the developing world.

Biomarkers for PAM have been challenging to identify because little is known about N. Fowleri's pathophysiology. Mass spectrometry, NMR and other tools of analysis are being utilized to identify biomarkers. Researchers are also making biochemical profiles of individuals in populations that contracted the disease against those who did not. These profiles will include information on the individual's age, gender, ethnicity and factors that predispose them to this condition³. This condition is rare and because of its rarity has been studied infrequently. With limited time and resources N.Fowleri is funded and studied less frequently than conditions that affect larger populations like Malaria or the Zika virus.

The range of drugs used to treat patients with PAM is severely limited, researchers are developing drugs that would inhibit different processes of N.Fowleri. The drug pathways are "hydrolytic enzymes that invade the host cells, glycocytic enzymes that are expressed differently by the pathogen, thiol based redox metabolism pathway, oxidative stress pathway, trypanothione pathways, and encystation and excystation pathways"³.

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