Naegleria fowleri (N. fowleri), commonly known as the brain-eating amoeba, causes primary amebic meningoencephalitis (PAM). This rare and fatal microorganism is found in freshwater. It enters the human body through the nasal passage, migrating into the brain, which causes severe inflammation and destruction of brain tissue. Over the past 50 years, 300 cases have been reported globally, with Pakistan reporting a notably higher number of cases, mainly attributed to humidity. Notably distinct age group susceptibility and infectivity patterns have been observed in Pakistan, particularly in Karachi, where ground saline water is consumed as the main source, suggesting the emergence of a new adaptable strain. PAM presents symptoms like bacterial meningitis, complicating diagnosis. An official diagnosis is made using cerebrospinal fluid (CSF) analysis, which shows polymorphonuclear leukocytes and trophozoites from N. fowleri. Treatment combines drug therapy and supportive care. Genotyping, raising awareness, clinical trials, and better water sanitation are vital to mitigate N. fowleri’s impact. This review highlights genetic diversity, improved diagnostics, and effective treatments.

Naegleria fowleri, Brain-eating amoeba, Primary amoebic meningoencephalitis, Freshwater pathogen, Amphotericin B, Cerebrospinal fluid, Olfactory tract.

The pathogen N. fowleri, also called brain-eating amoeba, was found to be the only pathogen responsible for PAM [1]. N. fowleri is a member of the kingdom Protista, phylum Amoebozoa, class Tubulinea, and family Vahlkampfiidae [2]. The life cycle of this pathogen has three stages: a cyst, a flagellate form, and an amoebic form; the latter is responsible for causing PAM. These species are found in air and soil and mainly in water sources, especially groundwater sources, which are the largest natural reservoirs for this pathogen [3,4]. To date, there are only 47 species in the Naegleria genus, of which  N. australiensis, N. italica, and N. fowleri have been described as pathogenic. Among the species of N. fowleri, it comprises eight distinct species, distinguished by variations in the length of their Internal Transcribed Spacers 1 sequences (ITS 1) and alterations in nucleotide arrangements [5]. Regarding geographical distribution, N. fowleri is unequally distributed throughout the world except Antarctica, especially in hot climates [6].

Genotypes 1, 2, and 3 are present in America, genotypes 2, 3, 4, 5, 6, 7, and 8 are harbored in Europe, and genotypes 2 and 3 exist in Asia. Among these genotypes, only genotypes 1, 2, 3, and 5 are pathogenic [7]. Worldwide, about 300 cases of N. fowleri have been reported in the past 50 years, with the first being reported in 1965 [8].

High-temperature climates like Pakistan are particularly susceptible to the occurrence of PAM. In 2008, the first recorded instance of PAM in Pakistan was identified in a young boy from Karachi who had a prior background of swimming in a river. By 2019, the number of reported PAM cases in Karachi alone reached 146. Remarkably, within just a decade, the incidence of PAM cases in Pakistan exceeded that of the United States of America, where 142 cases were reported between 1968 and 2019 [9]. Given Karachi’s heavy dependence on saline groundwater as its water source [10], the incidence of N. fowleri in the city is indeed intriguing. It is noteworthy that N. fowleri is typically unable to survive in saline water and primarily thrives in freshwater environments [11]. This contradiction raises the possibility of a new strain or variant of the pathogen capable of adapting to different water conditions, potentially contributing to the prevalence of cases in this region. Further investigation is necessary to explore this possibility and gain a better understanding of the pathogen’s behavior and characteristics.

Epidemiology
N. fowleri cases have been reported in multiple cities across Pakistan, including Quetta, Lahore, and Karachi. In 2023, the reported cases include four from Karachi, one from Quetta, one from Lahore, and one from Hyderabad. Notably, Karachi has consistently reported the most cases since the first instance of PAM documented in 2008 [1]. The province of Punjab, specifically Lahore, also experienced a few cases, with the initial case reported in 2015 [2,3].

Furthermore, strains of N. fowleri have been isolated in the Kashmir province; however, no cases of PAM have been documented in this region [4]. Table 1 in the text provides a comprehensive overview of how the incidence of PAM cases has varied since 2008 [1].

Table 1: Incidence of PAM cases from 2008 to 2023

Life Cycle
N. fowleri, an amoeba that exists in freshwater reservoirs like lakes, ponds, rivers, and groundwater sources such as wells, undergoes three distinct phases in its life cycle.

• Cyst stage
• Trophozoite stage (reproductive and infective stage)
• Flagellated phase

When the environment provides favorable conditions, N. fowleri thrives in its trophozoite stage and divides by promitosis [42]. However, when the availability of food sources becomes inadequate, the pathogen transitions from the trophozoite stage to the flagellated phase. In situations where the environment becomes entirely unfavorable, the trophozoite transforms into a cyst, which is capable of surviving within a wide temperature range, from above freezing to 65 degrees Celsius [44].

An important aspect to note about the life cycle of N. fowleri is that the trophozoite stage is the one responsible for infecting humans and animals during recreational water activities. The pathogen particularly resides in warm fresh water and hence, can be acquired by any activity involving contact of water with the human nasal passages [12]. Consequently, the trophozoite form is typically observed in CSF and tissue analysis. While flagellated forms may occasionally be present in the CSF, it is uncommon for cysts to be associated with brain tissue infections [42].

Pathogenesis
N. fowleri, also known as the brain-eating pathogen, is the cause of primary amoebic meningoencephalitis [12]. The presence of amoeba in the nasal mucosal and submucosal plexus and in the brain was demonstrated by light and electron microscopy. It was postulated that the route adopted by the pathogen begins with the nasal mucosa and the olfactory nerve cells, and from there the pathogen invades the olfactory bulb via the nerve bundles, passing through the openings in the cribriform plate of the ethmoid bone [13]. The olfactory bulb is connected to the olfactory tract, eventually leading the microbe to the inferior temporal lob, also known as the olfactory area of the brain [14].

The proposed mechanism for the eventual development of meningoencephalitis is the destruction of the neuronal cells by the amoeba’s lytic enzymes, such as hydrolases, phospholipases, and neuraminidases, combined with the initiation of a robust immune response by the host [15]. The release of cellular chemicals and components recruits macrophages and neutrophils to further activate the inflammatory cascade. The cellular debris is ingested by N. fowleri.

Another mechanism suggested by the researchers involves the ‘food cup theory’. Food cups refer to surface matter expressed by the pathogen, which allows it to ingest bacteria, fungi, and possibly human cells [15]. These mechanisms trigger an inflammatory response in the brain tissue, damaging it and the meninges, hence presenting with fulminant necrotizing meningoencephalitis.

Genotype
When discussing the genetic makeup of N. fowleri, the Internal transcribed spacer (ITS) and 5.8 recombinant deoxyribonucleic acid (rDNA) between the small subunit (SSU) and large subunit (LSU) rDNA are essential for differentiation. Type 1 and type 2 strains of N. fowleri have a similar ITS1 sequence of 46 base pairs (bp). The only distinguishing factor between them is a cytosine (C) to thymine (T) transition at position 31 in the rDNA. Similarly, types 3 and 4 have an identical ITS1 sequence of 142 bp. The key differentiation lies in a C to T transition at position 31 in the rDNA. The Type 5 genotype of N. fowleri interestingly shows a difference in the ITS 1 compared to other genotypes, with T present at position 31 of the rDNA sequence. Types 6, 7, and 8, on the other hand, have different ITS 1 sequences, having no C at position 31 of rDNA [1]. It should also be noted that among the genotypes mentioned, 1, 2, 3, and 5 have been found to be clinically significant. A case of N. fowleri, diagnosed at Imam Clinic in Karachi, Pakistan, has undergone genotyping. PCR-based analyses of the patient’s CSF and residential tap water samples, utilizing Naegleria species-ITS and N. fowleri-specific primers, uncovered the presence of N. fowleri DNA. The nucleotide sequences of the ITS primer-based amplicons from the patient’s CSF and water samples have been deposited in GenBank. Notably, phylogenetic analysis revealed that the N. fowleri isolates from Pakistan exhibited the type 2 genotype, indicating its position as one of the more recent evolutionary developments within the N. fowleri lineage. This finding contrasts with the genotypes observed in cases from the USA and the Philippines, which display a considerably more ancestral form of the pathogen [7].

Moreover, in 2019, a novel strain of N. fowleri, designated as Karachi-NF001, was discovered in a patient in Karachi. Notably, the genome of Karachi-NF001 revealed 15 distinct genes not identified in any previously reported N. fowleri strains worldwide. Among these, six genes were found to encode widely recognized proteins.

Nevertheless, it is essential to note that the genomic characterization of N. fowleri in Pakistan remains relatively limited, as the cases described above represent limited data. Consequently, further comprehensive investigations are imperative to gain a more holistic understanding of the genetic diversity within the region’s N. fowleri population.

There has not been much research on the difference in presentation of N. fowleri depending on its genotype. A literature review on the global presentation of N. fowleri has shown fever to be the most common presentation among patients, followed by headache, nausea/vomiting, altered mental status, and nuchal rigidity. Of the cases reported in the review, most of them had a history of recreational water activities and a median age of 14 years [16]. When comparing these variables to the cases in Pakistan we find the median age for presentation is 29.47 years (standard deviation [SD]: 13.95), a case fatality rate of 98% compared to the global fatality rate of 92%; most of cases present with fever, headache and neurological manifestations [17,18]. Among all the documented cases of PAM in Pakistan, exclusively Muslims were affected, and only two individuals had a background involving recreational water activities. The available information suggests a potential presence of N. fowleri in the domestic water supply, indicating a possible association with ablution practices [19]. A case series of PAM diagnosed via PCR and/or wet preparation examination of CSF conducted in 2017 at the Aga Khan University Hospital located in Karachi, Pakistan, reported that out of the 116 CSF specimens collected, only 19 were positive for N. fowleri, among those 16 (84%) were male and 3 (16%) were female [12,20]. Despite the difference between genotypic variants of N. fowleri found around the globe and in Pakistan, the clinical presentation is similar, except for the distinguishing age preference of the variant found in Pakistan.

Considering the high mortality rate associated with PAM, the inclusion of PAM as a possible cause of any patient presenting with symptoms as stated above is of prime importance. The similarities between the clinical presentation of PAM and bacterial meningitis render diagnosis on clinical grounds difficult. Hence, keeping PAM as one of the differentials, prompt measures would include baseline assessment (complete blood count, comprehensive metabolic profile, Prothrombin time, Activated partial thromboplastin time) computerized tomography (CT)/magnetic resonance image (MRI), lumbar puncture, CSF analysis, microscopic examination, serology and polymerase chain reaction (PCR) [21].

In the early phases of the infection, MRI imaging and contrast-enhanced CT scans typically show signs of compression of the brain’s surface folds (cortical sulcal effacement), swelling of the cerebrum, and the filling of spaces around the subarachnoid region and the midbrain. As the infection advances, these signs usually deteriorate, leading to the emergence of necrotic regions, the formation of aneurysms, and the narrowing of blood vessels (stenosis). A reverse transcription PCR (RT-PCR) is also indicated to establish the genus and species of the amoeba [21]. The diagnosis of PAM must be officially determined via CSF from a lumbar puncture. The CSF is examined using microscopic methods such as amebic antigen detection, CSF wet film preparation, and CSF culture. It frequently contains many polymorphonuclear leukocytes and trophozoites from N. fowleri. The CSF sample must be stained with Giemsa-Wright or trichrome to detect and visually study the amoeba [22,23]. Additionally, immunofluorescence (IF) assay, flow cytometry (FC), enzyme-linked immunosorbent assay (ELISA), and microscopy can be further employed to verify the presence of amoeba [22]. In most of the patients, intracranial and CSF pressures were found to be elevated, sometimes as high as 600 mm H2O or more, an indicator that has been strongly linked to the patient’s mortality. This heightened pressure is often attributed to the cause of death, involving increased intracranial pressure leading to brain herniation, ultimately resulting in cardiopulmonary arrest and pulmonary edema. CSF may be gray to yellow white in the initial phases of the disease, but as the condition advances, it can become red due to the enormous quantity of red blood cells (RBCs), which has been recorded as high as 24,600/mm3 [15].

In the survey carried out at Aga Khan University Hospital, Karachi, Pakistan, among the 116 CSF samples collected, PAM was associated with an elevated number of neutrophils, increased proteins, and decreased concentrations of glucose [20]. A global review analyzing 381 PAMCSF samples reported similar findings [16]. According to the statistics, 156 cases of N. fowleri were reported in the United States, 41 in Pakistan, 33 in Mexico, and 22 in Australia in a span of 81 years (see table 2).

Table 2: Reported cases of PAM, 1937–2018, by country (n=381) [25]

Although PCR-based procedures used at institutions such as Aga Khan University Hospital, Karachi, Pakistan, are extremely successful, they have certain downsides, such as costly expenditures. Furthermore, product amplification and identification need advanced technology and competent laboratory staff. As a result, these characteristics make PCR inappropriate for application in impoverished nations or resource-constrained countries such as Pakistan [26]. In addition, instances of PAM are sometimes incorrectly labeled as meningitis caused by bacteria due to a lack of awareness regarding N. fowleri among health professionals and the public, which leads to delayed and improper treatment [20].

Based on mass spectrometry, new techniques such as matrix-assisted laser-desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF MS) can be used to detect N. fowleri quickly by detecting peptides and small acidic proteins, desorbed from whole cells. MALDI-TOF MS is an efficient and rapidly advancing technique for swiftly detecting microorganisms and distinguishing between different strains [27]. The first ever MALDI-TOF MS system in Pakistan was installed at Chugtai Lab, Lahore, by the name of VITEK MS in 2019 [28]. A highly sensitive and specific loop-mediated isothermal amplification (LAMP) technique was developed to identify N. fowleri. LAMP offers advantages such as simplicity, affordability, and visual result interpretation [29]. It showed excellent specificity and sensitivity in detecting even small amounts of N. fowleri DNA or intact amoebae in samples of water, including flood water, which makes it useful as a diagnostic tool for N. fowleri in field settings; further validation using clinical specimens is needed [26].

Since the pathogen is not very common, there is no definitive treatment. However, various clinical trials have been conducted in the past, which showed certain medications to have acceptable though not promising efficacy against the pathogen. The approved set of drugs includes Amphotericin B, fluconazole, azithromycin, rifampicin, miltefosine, and dexamethasone, with miltefosine being the most recent one [30]. The treatment strategy proposed to avoid devastating neurologic complications includes a combination of drug therapy initiated at the earliest possible time, maintaining cerebral blood flow, intracranial pressure, and respiratory rate [31]. Among the antifungal drugs, Amphotericin B is the most commonly used drug, which can be administered intravenously or intrathecally (maximum IV dose of 1.5 mg/kg/day; maximum intrathecal dose of 1.0 mg/day), either as a single drug or in combination with other approved drugs such as rifampicin or azithromycin. The antimicrobial action of Amphotericin B is primarily by forming pores in the pathogen’s cell membrane, thereby promoting apoptosis [32]. Miltefosine, an antileishmanial agent, became a hope to finally tackle the deadly pathogen when a 12-year-old female survived PAM without neurological deficits following miltefosine therapy [33]. However, the other two cases of a 12-year-old male and an 8-year-old male suffering from meningoencephalitis due to N. fowleri, which received a similar treatment regimen involving miltefosine and aggressive supportive management, showed different outcomes. Of these two cases, the 12-year-old boy was eventually proven brain dead, and the 8-year-old survived but with significant neurological deficits [34]. CDC recommendations include intravenous fluconazole at a dose of 10 mg/kg/day once daily (maximum dose of 600 mg/day) for 28 days and intravenous azithromycin at doses of 10 mg/kg/day once daily (maximum 500 mg daily dose) for 28 days as adjunctive medications [35]. However, additional clinical trials are still required to establish the effectiveness and safety of the mentioned drugs, as well as to establish a conclusive treatment approach with appropriate dosage levels within the effective and non-toxic ranges.

Also, massive inflammation in the brain causes cerebral edema, which elevates the intracranial pressure, which in turn predisposes the infected ones to develop brain herniation. Research-proven data suggests the implication of monitoring hypothermia (32- 340C) in the management of encephalitis as it helps in halting inflammation by lowering cytokines, leukocyte response, and nitric oxide, thereby also decreasing cerebral edema [36]. The chances of complete recovery depend on early diagnosis, immediate initiation of the treatment plan, vigilant monitoring, and prompt symptomatic management.

Prevention
Warm and humid climate promotes the growth of this amoeba; in Pakistan, climatic change and subsequent global warming have led to extended summers as well as increased humidity, specifically in coastal regions [37]. From 2002 to 2006, the Pakistan Council of Research in Water Resources (PCRWR) undertook an extensive investigation into water quality across 23 major cities spanning all four provinces of the country. The study’s results reveal that, on average, 84-89% of water sources across Pakistan do not meet the recommended safety standards for human consumption. Surface and groundwater are significant water sources for the people of Pakistan, and it is noteworthy that the majority of documented N. fowleri cases were concentrated in Karachi, a city heavily dependent on groundwater [38,39].

Upon retrospectively reviewing the cases of N. fowleri in Pakistan, it is notable that none of the patients had a documented history of engaging in recreational water activities. Instead, the cases were found to be linked to ablution [40]. Research has established that Naegleria trophozoites and cysts are susceptible to chlorine and monochloramine [41]. This knowledge presents valuable opportunities for effective prevention strategies. For instance, when swimming in pools during the summer season, it is advisable to ensure proper chlorination to minimize the risk of Naegleria infection and limit recreational water activities during summer. Boiled water is recommended for ablution, but in situations where it is unavailable, distilled or sterile water can be used. Since pre-treating the water with chlorine halts the survival of the pathogen, it is highly recommended that households supply chlorinated water [42]. However, the rising trend of Naegleria cases observed in Pakistan questions the credibility and safety of the water and sewerage board supply [43]. Therefore, successful prevention can only occur if the higher authorities incorporate efficient methods to deal with and ameliorate the water supply management of the city.

In recent years, a concerning trend has emerged in Pakistan, indicating a significant increase in N. fowleri cases. Remarkably, Pakistan has exceeded the reported cases in the United States of America within a fifty-year timeframe. Notably, N. fowleri demonstrates distinct variations in terms of age group susceptibility, mortality rates, and infectivity within Pakistan compared to the global scenario. Moreover, the tendency of N. fowleri was reported to be highest among people living near the coastal areas whose population depends on saline water, suggesting that the genotype in Pakistan may belong to a different strain. Consequently, genotyping and raising awareness about this pathogen have become crucial in tackling this issue effectively. Due to the global rarity of this infection, there has been considerably less research on the management protocols. The available treatments have not produced promising results, and hence, further trials are required to devise a reliable and definitive treatment plan.

Considering the mortality associated with the infection, devising prompt measures might help in lowering the high death rate, which can be achieved by conducting further drug trials. Misdiagnosis can also be a contributing cause of higher death rates in Pakistan, likely due to a lack of resources, a lack of awareness among clinicians, as well as a similar presentation of PAM with bacterial meningitis. Muslims practicing ablution are at an increased risk of N. fowleri infection. With a dense Muslim population in Pakistan, the risk is amplified. Thus, it is vital to prioritize a clean, chlorinated water supply, especially in vulnerable cities, as current water standards in Pakistan fall below global norms. Additionally, it is worth noting that N. fowleri predominantly affects a specific age group, primarily middle-aged to young adults, in Pakistan. This raises questions regarding the apparent lack of infection among older individuals. One possible explanation could be that the amoeba encounters a less favorable environment in the bodies of older individuals. Nevertheless, further research is needed to gain deeper insights into this matter.

We are deeply grateful to all those who played a role in gathering and drafting this manuscript. All the authors contributed significantly in multiple ways.

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Corresponding Author: 
Raja Devendar
Department of Community Medicine
Dow University of Health Sciences, Dow Medical College, Karachi, Pakistan
Email: rajadevinder97@gmail.com

Co-Authors:
Abbas Mustafa Gain, Rabeea Mushtaq, Ashraf Jahangeer
Department of Community Medicine
Dow Medical College, Dow University of Health Sciences, Karachi, Pakistan

Naseha Mushtaq
Department of Medicine
Karachi University, Karachi, Pakistan

Rafay Shahab Ansari
Department of Medicine
Ziauddin Medical College, Ziauddin University, Karachi, Pakistan

All authors contributed to the conceptualization, investigation, and data curation by acquiring and critically reviewing the selected articles. They were collectively involved in the writing – original draft preparation, and writing – review & editing to refine the manuscript. Additionally, all authors participated in the supervision of the work, ensuring accuracy and completeness. The final manuscript was approved by all named authors for submission to the journal.

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https://doi.org/10.63096/medtigo3062247

Abbas Mustafa G, Rabeea M, Raja Devendar, Naseha M, Ashraf J, Rafay Shahab A. Naegleria Fowleri: Insights and Emerging Strain in Pakistan. medtigo J Med. 2024;2(3):e3062247. doi:10.63096/medtigo3062247 Crossref