The Tale of Naegleria fowleri and Primary Amoebic Meningoencephalitis

By Kenneth Chan

Abstract

Primary amoebic meningoencephalitis (PAM) is a destructive brain disease caused by the thermophilic free-living amoeba, Naegleria fowleri. One could get infected by this amoeba when exposed to warm, fresh water, typically from rivers and lakes. The amoeba enters the host through the nasal cavity during recreational water activities. PAM has an incredibly high mortality rate due to its being hard to diagnose, its fast progression, and rarity. Targeted diagnostic tests can identify the disease and treatment is available, but a prompt diagnosis and treatment are required to successfully cure the disease.

Introduction

PAM is a lethal infectious disease of the central nervous system (CNS) that affects mostly healthy children and young adults. PAM is a rapidly progressive disease that has a fatality rate of greater than 97%. The causative agent of the disease is Naegleria fowleri, a thermophilic, free-living amoeba that is most commonly found in warm, fresh water such as ponds, lakes and rivers (Capewell et al., 2015). There are over 40 species of Naegleria, but PAM is induced only by an infection with N. fowleri (Wang et al., 2018). Infection with N. fowleri can occur when an individual is exposed to contaminated water, e.g. through recreational water activities such as swimming or diving, where the amoeba enters the body via the nasal cavity. Amoebas will first attach to the host’s nasal mucosa and then migrate along the olfactory nerve to reside in the olfactory bulb within the CNS (Chen et al., 2019).

Parasite Biology

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*Figure 1. Naegleria fowleri morphology: (a) trophozoite (b) flagellate (c) cyst. All images at x1000 magnification.
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*Figure 2. Scanning electron micrograph of trophozoites ‘food-cups’.

Naegleria species take three morphological forms throughout their life cycle, and are able to switch between trophozoite, flagellate, and cyst forms (Fig. 1). The trophozoites are able to reproduce via binary fission. Trophozoites feed on bacteria and yeast in the environment using ‘food-cups’ on their surface (Fig. 2) (Marciano-Cabral & Cabral, 2007). They also display hemispherical bulges, called lobopodia, which allow active locomotion. While trophozoites are presumed to be the infectious form for humans, it is plausible that cysts are able to enter the host and then switch to their trophozoite form and invade the CNS. When the trophozoite is exposed to nutrient-depleted conditions, or experiences ionic fluctuation, it is able to transform into a flagellate for (Visvesvara et al., 2007). This flagellate form lasts for an hour before reverting back to the trophozoite form. It will not be able to feed, or undergo binary fission, at this stage. As soon as the food supply diminishes, or the trophozoite is exposed to adverse conditions, it will transform into the resistant cyst. It usually takes a spherical double-walled shape (Visvesvara, 2010).

Epidemiology

The first case of PAM was reported by Fowler and Carter (1965) in Australia. Despite there being cases of PAM in several countries, it is believed that N. fowleri is distributed worldwide. The United States has the highest PAM incidence, documenting 138 cases over 50 years, averaging between zero and eight cases annually (Cope & Ali, 2016). A review of cases reported in 2008 in the United States revealed that PAM generally occurs in previously healthy young males that have been exposed to warm, recreational waters, in the southern states during the summer period (Yoder et al., 2010).

In recent years, there are a few notable changes in the distribution and incidence of PAM. There are cases of people contracting PAM within northern states in the United States. One case was reported in Minnesota in a patient with no history of leaving the state (Kemble et al., 2012). An environmental investigation was done on the lake in which the patient swam, where it tested positive for N. fowleri. Since 2010, there has been an increasing number of PAM cases reported in northern states, including Kansas and Indiana. Considering the influence of climate change, the growth in PAM cases outside southern states are of great concern and it suggests that physicians should consider PAM as part of their differential diagnosis in other states.

In addition to the changed PAM distribution, changes have also been reported in the route of transmission. A review highlighted that 80% of PAM cases arose from exposure to natural water, however, slightly less than 2% of cases resulted from exposure to tap water (Yoder et al., 2010). It was later revealed that these patients were regular users of neti pots for nasal irrigation, and that water samples taken from their houses tested positive for N. fowleri (Yoder et al., 2012). N. fowleri trophozoites and cysts are rendered inactive at chlorine concentrations of 0.5 mg/L, and hence these levels have been adopted into swimming pools to mitigate the risk of infection (Chang, 1978).

Clinical Presentation

Physicians encountering immunocompetent children and young adults that have had recent exposure to fresh water, and whom display acute neurological symptoms as described below, should consider PAM as part of their differential diagnosis. In the early stage of the infection, generic symptoms of high fever, sudden onset of bifrontal or bitemporal headache, and nuchal rigidity are common.  Onset to illness from exposure is between 5-7 days, but it can be as short as 24 hours. Due to the absence of a hallmark clinical feature, it is difficult to separate PAM from acute pyogenic or bacterial meningoencephalitis. Hence, it is critical that the attending physician identifies any freshwater contact made in the past week. Primary signs are usually accompanied by nausea, vomiting, and irritability. When nuchal rigidity occurs, both Kernig’s and Brudziński’s signs – signs of meningitis which are indicated by pain in the knee when it is lifted at a right angle (Kernig’s sign), and by forearms flexing reflexively after pressure is applied on the cheek (Brudziński’s sign) – are common.

During later stages, neurological symptoms such as seizure, confusion, diplopia or coma will be present (Grace et al., 2015). An increase in intracranial pressure is expected during the course of the disease. Cranial nerve palsies should be noted as signs of brain oedema and herniation. The main cause of death in PAM is increased intracranial pressure, coupled with herniation of the brain, which ultimately leads to cardiopulmonary arrest and pulmonary oedema (Capewell et al., 2015). The disease progresses rapidly, with the median time from onset of symptoms to death is 5 days. This rapid progression emphasises the importance of prompt diagnosis and intervention. PAM is not considered often in differential diagnosis, as the disease is rare and can be misdiagnosed as bacterial meningoencephalitis. By the time PAM is considered and treated for, it is often too late for the patient and would leave them with severe neurological deficit or even death. To distinguish between the two diseases, PAM must be specifically tested for, as described below.

Diagnostic Testing

Collection of Clinical Specimens

In order to test for PAM, specimens must first be obtained from the patient. CSF or brain tissue are essential specimens for diagnosis. These can be retrieved via a lumbar puncture for CSF, or a biopsy for brain tissue. The former method is preferred as it is less invasive and has a lower probability of complications such as strokes and seizures. Specimens should be collected aseptically and stored at room temperature prior to any laboratory examination. These conditions favour the survival of the amoeba for direct observation, allowing growth within culture media. When manipulating the samples, appropriate personal protective equipment (PPE) should always be worn and samples should be contained in biological safety cabinets (Cope & Ali, 2016).

Microscopic Identification

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*Figure 3. Cytospin of fixed CSF, stained with Giemsa-Wright,  showing a Naegleria fowleri trophozoite (arrow) amidst polymorphonuclear leukocytes and a few lymphocytes. Within the trophozoite, the nucleus and nucleolus can be seen. 1000x magnification.

A wet mount of the CSF sample is examined for the presence of motile trophozoites. When the samples are ready to be examined, the sample container should be gently agitated in order to dislodge any amoebas that may have adhered. The sample should be centrifuged and the supernatant removed, leaving 200-300 mL of residual liquid. Samples are incubated at 35-37°C before being treated with a differential stain. Giemsa or Wright stains are used on the smears of the CSF, allowing host cells to be distinguished from amoebas by a large, central nucleolus (Capewell et al., 2015).

This method is a fast, simple, and cheap preliminary diagnostic test. However, trained personnel are required to accurately separate amoebas and host cells. However, this examination may return false negatives, as it is dependent on the morphological integrity of the amoebas in the specimen. A confirmational test is required to effectively rule out N. fowleri infection (Cope & Ali, 2016).

Molecular Detection

Many molecular techniques have been developed for the precise identification of N. fowleri in both clinical and environmental samples. These techniques usually employ detection by a polymerase chain reaction (PCR) or isothermal DNA amplification. Three targets for PCR detection are currently being exploited: the mitochondrial 5.8S and 18S rRNA sequences, and the internal transcribed spacers region. These assays share similar sensitivities and specificities when employed to detect either the Naegleria genus, or specifically N.fowleri (Streby et al., 2015).

The most popular method that is currently used to test clinical specimens is the TaqMan real-time PCR assay. This test is perfect for PAM detection as it takes only 2-3 hours, addressing the time-sensitive nature of PAM diagnosis. Amoebas contain many copies of the target sequence, giving this method incredible sensitivity (Qvarnstrom et al., 2006). The test is highly specific to N. fowleri and does not replicate the DNA of other amoebas, such as Acanthamoeba castellanii or Balamuthia mandrillaris, which are attributed to granulomatous amoebic encephalitis (GAE).

Real-time PCR is regarded as the ‘gold standard’ for the diagnosis of suspected PAM cases. Whilst this test is sufficient if deployed appropriately, new techniques are being developed for the unbiased detection of multiple pathogens simultaneously. Metagenomic next-generation sequencing (mNGS) is an exciting method that offers such an advantage. It serves as a powerful tool  in the rapid identification of known and unknown pathogens, rather than relying on the hypothesis of the attending physician (Gu et al., 2019). mNGS has been used successfully in China for the rapid diagnosis of PAM, however, the patient succumbed to the disease due to its rapid progression (Wang et al., 2018). The technique is not perfect, however, as the host genome needs to be sequenced to eliminate background. This may be countered by the increasing speed of human genome sequencing, particularly in the clinical setting, allowing mNGS to be widely adopted for the diagnosis of PAM and many other diseases.

Treatment

As N. fowleri infections are rare, there are no clinical trials to date that assess the efficacy of each treatment regimen. The majority of the information used for treatment is gathered from either case reports or in vitro studies. Despite the absence of a standardised regimen, the use of amphotericin B is the most common first line of treatment, delivered either intravenously or intrathecally. It is shown that amphotericin B induces apoptosis-like programmed cell death in Naegleria spp.The exact mechanism for this sensitivity in Naegleria is still unknown (Cárdenas-Zúñiga et al., 2017). Amphotericin B is deemed a critical drug in the treatment of PAM attributed to N. fowleri and can be used with or without other adjunctive therapies as a recommended treatment for 10 days. Other adjunctive therapies include the use of rifampin or an azole drug (miconazole or fluconazole). Miconazole is of particular interest, as it is believed to have a synergistic effect with amphotericin B (Grace, et al., 2015). Steroids, such as dexamethasone, are also given in order to control the cerebral oedema caused by PAM. Full recovery from PAM is noted when following these recommendations, however, delays in treatment can result in the patient having lingering neurological deficits, or succumbing to the damage done by the disease (Grace, et al., 2015).

Conclusion

PAM is an incredibly lethal CNS infection that one can get upon exposure to contaminated, warm freshwater. Despite the fact that this is a very rare condition, and that most physicians may never encounter it, it should be considered in the differential diagnosis, as appropriate intervention is crucial to prevent an almost certain fatal outcome. There are many diagnostic techniques readily available to aid in the process, with mNGS promising to be a useful tool. Further research is needed to assess the efficacy of potential treatment regimens, which could prove invaluable in reducing the mortality of PAM.

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