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Naegleria fowleri (True Pathogen – Primary Amebic Meningoencephalitis/PAM)

Organism:
Infections of the central nervous system (CNS) caused by free‑living amebae have been recognized only since the mid‑1960s, and our understanding of this disease process is still incomplete. One type of meningoencephalitis (PAM) is a fulminant and rapidly fatal disease that affects mainly children and young adults. The disease closely resembles bacterial meningitis but is caused by N. fowleri, an organism found in moist soil and freshwater habitats. Close to 200 cases of PAM have occurred worldwide, and approximately 90 of those cases have been reported from the United States. Until recently, it was believed that this infection was limited to humans; however, infections have also been reported in other animals.

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Naegleria fowleri trophozoites Trophozoite Cyst from culture

 

Trophozoite wet mount Flagellated stage

 

Naegleria trophozoites in tissue (cysts are not seen in tissue)


Life Cycle:
The trophozoites can occur in two forms, ameboid and flagellate. Motility can be observed in hanging‑drop preparations from cultures of cerebrospinal fluid (CSF); the ameboid form (the only form recognized in humans) is elongate with a broad anterior end and tapered posterior end. The size ranges from 7 to 35 μm. The diameter of the rounded forms is usually 15 μm. There is a large, central karyosome and no peripheral nuclear chromatin. The cytoplasm is somewhat granular and contains vacuoles. The ameboid‑form organisms change to the transient, pear‑shaped flagellate form when they are transferred from culture or teased from tissue into water and maintained at a temperature of 27 to 37°C. The change may occur very quickly (within a few hours) or may take as long as 20 h. The flagellate form has two flagella at the broad end. Motility is typical, with either spinning or jerky movements. These flagellate forms do not divide, but when the flagella are lost, the ameboid forms resume reproduction.
Cysts from nature and from agar cultures look the same and have a single nucleus almost identical to that seen in the trophozoite. They are generally round, measuring from 7 to 15 μm, and there is a thick double wall.

Acquired:
The amebae may enter the nasal cavity by inhalation or aspiration of water, dust, or aerosols containing the trophozoites or cysts. The organisms then penetrate the nasal mucosa, probably through phagocytosis of the olfactory epithelium cells, and migrate via the olfactory nerves to the brain. Data suggest that N. fowleri directly ingests brain tissue by producing food cups or amebostomes, in addition to producing a contact‑dependent cytolysis which is mediated by a heat‑stable hemolytic protein, heat‑labile cytolysis, and/or phospholipase enzymes. Cysts of N. fowleri are generally not seen in brain tissue.

Epidemiology:
Nearly 200 presumptive or proven cases of PAM have been reported in the literature, including cases from the United States, Ireland, England, Belgium, Czechoslovakia, Australia, New Zealand, Brazil, and Zambia. Clinical patient histories indicate exposure to the organism via freshwater lakes or swimming pools (not in the US due to chlorination) shortly before onset; patients had been previously healthy with no specific underlying problems. Pathogenic Naegleria organisms have also been isolated from nasal passages of individuals with no history of water exposure, thus suggesting the possibility of airborne exposure.

Clinical Features:
Early symptoms include vague upper respiratory distress, headache, lethargy, and occasionally olfactory problems. The acute phase includes sore throat, stuffy blocked or discharging nose, and severe headache. Progressive symptoms include pyrexia, vomiting, and stiffness of the neck. Mental confusion and coma usually occur approximately 3 to 5 days prior to death. The cause of death is usually cardiorespiratory arrest and pulmonary edema.
PAM can resemble acute purulent bacterial meningitis, and these conditions may be difficult to differentiate, particularly in the early stages. The CSF may have a predominantly polymorphonuclear leukocytosis, increased protein, and decreased glucose concentration like that seen with bacterial meningitis.  Unfortunately, if the CSF Gram stain is interpreted incorrectly (identification of bacteria as a false positive), the resulting antibacterial therapy has no impact on the amebae and the patient will usually die within several days.

Clinical Specimen:
CSF:  Spinal fluid placed on a slide with coverslip (do not use counting chamber – organisms will look like WBCs).

Laboratory Diagnosis:  THIS REQUEST IS ALWAYS A STAT!
Specimens should never be refrigerated prior to examination. When centrifuging the CSF, low speeds (250 ´ g) should be used so that the trophozoites are not damaged. Although bright‑field microscopy with reduced light is acceptable, phase microscopy, if available, is recommended. Use of smears stained with Giemsa or Wright's stain or a Giemsa‑Wright's stain combination can also be helpful. If N. fowleri is the causative agent, trophozoites only are normally seen. If the infecting organism is Acanthamoeba spp., cysts may also be seen in specimens from CNS infection. Unfortunately, most cases are diagnosed at autopsy; confirmation of these tissue findings must include culture and/or special staining with monoclonal reagents in indirect fluorescent antibody procedures. Organisms can also be cultured on nonnutrient agar plated with Escherichia coli.
In cases of presumptive pyogenic meningitis in which no bacteria are identified in the CSF, the computed tomography appearance of basal arachnoiditis (obliteration of basal cisterns in the precontrast scan with marked enhancement after the administration of intravenous contrast medium) should alert the staff to the possibility of acute PAM.
The amebae can be identified in histologic preparations by indirect immunofluorescence and immunoperoxidase techniques. The organism in tissue sections looks very much like an Iodamoeba bütschlii trophozoite, with a very large karyosome and no peripheral nuclear chromatin; the organisms can also be seen with routine histologic stains.

Organism Description:
Trophozoite:  the ameboid form (the only form recognized in humans) is elongate with a broad anterior end and tapered posterior end. The size ranges from 7 to 35 μm. The diameter of the rounded forms is usually 15 μm. There is a large, central karyosome and no peripheral nuclear chromatin. The cytoplasm is somewhat granular and contains vacuoles. The ameboid‑form organisms change to the transient, pear‑shaped flagellate form when they are transferred from culture or teased from tissue into water and maintained at a temperature of 27 to 37°C. The change may occur very quickly (within a few hours) or may take as long as 20 h. The flagellate form has two flagella at the broad end. Motility is typical, with either spinning or jerky movements. These flagellate forms do not divide, but when the flagella are lost, the ameboid forms resume reproduction.
Cyst:  The cyst typically has a double wall and can be seen in culture, but not in tissue or the CSF.
Culture:  The most effective approach uses nonnutrient agar plates with Page's saline and an overlay growth of Escherichia coli on which the amebae feed. There is also evidence that phosphate‑buffered saline can be used. Specimens transported in ameba‑saline (5.0 ml) and filtered through 13‑mm, 0.22‑μm‑pore‑size cellulose acetate and nitrate filters (Millipore Corp., Bedford, Mass.) have also been acceptable for organism recovery. The filter is then placed in the center of the nonnutrient agar plate seeded with E. coli.  Tissue stains are also effective, and cysts can be stained with Gomori's silver methenamine, periodic acid‑Schiff, and calcofluor white.  Both cysts and trophozoites can be recovered from culture.

Laboratory Report:
Naegleria fowleri seen and/or cultured

Treatment:
Many antimicrobial and antiparasitic drugs have been screened for in vitro and in vivo activity against N. fowleri. Although N. fowleri is very sensitive to amphotericin B in vitro, only a few patients have recovered after receiving intrathecal and intravenous injections of this drug alone or in combination with miconazole. One case within California in which the patient was successfully treated with amphotericin B, miconazole, and rifampin has been documented. Delay in diagnosis and the fulminant nature of PAM result in few survivors; this is unfortunate since N. fowleri is quite sensitive to the antifungal agent Amphotericin B.
Garcia, L.S. 2007.  Diagnostic Medical Parasitology, 5th ed., ASM Press, Washington, D.C.

Control:
General preventive measures include public awareness of potential hazards of contaminated water. It has been recommended that warm discharge water not be used for sports and recreational purposes (20), particularly since DNA restriction fragment profiles of environmental strains and human isolates were homogeneous. N. fowleri cysts produced in the warm summer months may survive the winter and are capable of growth during the following summer.