Case Study
Naegleria fowleri That Induces Primary
Amoebic Meningoencephalitis: Rapid
Diagnosis and Rare Case of Survival
in a 12-Year-Old Caucasian Girl
Andrew L. Dunn, MD,1 Tameika Reed, MT (ASCP),2 Charlotte Stewart, MT (ASCP),2
Rebecca A. Levy, MD1,2*
Laboratory Medicine 47:2:149-154
DOI: 10.1093/labmed/lmw008
ABSTRACT
Primary amoebic meningoencephalitis (PAM) is a rare and almost
always fatal disease that is caused by Naegleria fowleri, a freshwater
thermophilic amoeba. Our case involves an adolescent female who
presented with fever of unknown origin. A lumbar puncture was
performed, and the Wright-Giemsa and Gram stained cerebrospinal
fluid (CSF) cytospin slides showed numerous organisms. Experienced
medical technologists in the microbiology and hematology laboratories
identified the organisms as morphologically consistent with Naegleria
species. The laboratory made a rapid diagnosis and alerted emergency
department care providers within 75 minutes. The patient was treated
for PAM with amphotericin, rifampin, azithromycin, fluconazole and
Primary amoebic meningoencephalitis (PAM) is a rare
disease of the central nervous system (CNS) that is almost
always fatal.1,2 This disease is caused by Naegleria fowleri, a
freshwater thermophilic amoeba. Infection is associated with
swimming and diving in freshwater lakes, hot springs, ponds,
and inadequately chlorinated pools and/or spas.3 The
amoeba enters the body through the nares, travels through
the nasal mucosa along the olfactory nerves, across the
cribriform plate, and enters the brain.4 Patients commonly
Abbreviations
PAM, primary amoebic meningoencephalitis; CNS, central nervous sys-
tem; CSF, cerebrospinal fluid; PCR, polymerase chain reaction; ED, emer-
gency department; CT, computed tomography; WBCs, white blood cells;
RBCs, red blood cells; CDC, Centers for Disease Control and Prevention;
MRI, magnetic resonance imaging
1
Department of Pathology, University of Arkansas for Medical Sciences,
Little Rock, 2Department of Pathology, Clinical Laboratory Arkansas
Children’s Hospital, Little Rock
*To whom correspondence should be addressed.
RALevy@uams.edu
C American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
V 149
Downloaded from https://academic.oup.com/labmed/article-abstract/47/2/149/2937887
by guest
on 25 August 2018
aggressive supportive therapy including dexamethasone. The Centers
for Disease Control and Prevention (CDC) was contacted, and
miltefosine, an investigational medication, was started. Additional
treatment included an intraventricular shunt and controlled
hypothermia in order to mitigate potential cerebral edema. Our patient
is a rare success story, as she was diagnosed swiftly, successfully
treated, and survived PAM.
Keywords: primary amoebic meningocephalitis, Naegleria fowleri,
thermophilic amoeba, microbiology, body fluid morphology, and body
fluid interpretation
experience fever, headache, nausea, vomiting, stiff neck,
and, occasionally seizures. N. fowleri and its associated
inflammatory reaction, including the release of cytotoxic
molecules, cause extensive tissue damage and necrosis.5,6
The sequelae include acute necrotizing meningoencephalitis
and usually result in death in 7 to 10 days. Other flagellates
found in the environment include several species of
Acanthamoeba and Balamuthia mandrillaris, both of which
can cause granulomatous amoebic encephalitis. It is
important to consider these entities when evaluating PAM.
Eight different types of the Naegleria fowleri pathogen show
an uneven distribution throughout the world; 3 of those types
have been identified within the United States.1,2 Two patients
in North America have been reported to have well-
documented treatments and survival despite contracting
PAM.7,8 The case of our patient, a 12-year-old proband girl
from Arkansas, represents the third case of reported survival.
The unique and experimental treatment plan for this
individual is well described in an article by Matthew Linam,
MD, an infectious disease specialist at Arkansas Children’s
 Case Study

Image 1
Naegleria fowleri on Wright-Giemsa stained cerebrospinal fluid cytospin slides (original magnification, 1000; oil immersion). Arrows indicate
Naegleria organisms. Specimen was donated by our patient, a 12-year-old Caucasian girl.

Hospital in Little Rock.9 However, the speed of the diagnosis
and the impact of this speed on the treatment algorithm have
not yet been described in the literature, to our knowledge.
PAM is a rare occurrence and is not often considered as a
likely diagnosis; therefore, the laboratory’s identification of
the microorganism may be the first time an amoebic etiology
is considered. This rapid identification can help avoid delays
in diagnosis and therapy.
Cytologic analysis of cerebrospinal fluid (CSF) in patients
with PAM typically shows an increase in neutrophils;
trophozoites of Naegleria can be identified in the CSF via
Wright-Giemsa stain.10,11 Earlier studies12,13 have shown
that CSF should be evaluated immediately after a lumbar
puncture procedure for best leukocyte survival. CSF is a
hypotonic solution, and leukocytes will lyse if left in the
medium; specifically, neutrophils lyse more rapidly than
lymphocytes and monocytes.12 Manual microscopy is the
criterion standard for bodily-fluid evaluation;14 assessment
of Wright-Giemsa and Gram stains also can be helpful, in
combination with patient history, in initiating a treatment
course for infectious disease while waiting for the results of
confirmatory cultures or polymerase chain reaction (PCR)
studies.

150 Lab Medicine 2016;47:2;149–154 www.labmedicine.com

150 DOI: 10.1093/labmed/lmw008
Downloaded from https://academic.oup.com/labmed/article-abstract/47/2/149/2937887
by guest
on 25 August 2018

Image 2
Naegleria fowleri on Gram-stained cerebrospinal fluid cytospin slides (original magnification, 500; oil immersion). Arrows indicate Naegleria
organisms. Specimen was donated by our patient, a 12-year-old Caucasian girl.
Case Presentation
A previously healthy 12-year-old Caucasian female girl
arrived at the emergency department (ED) with a 3-day
history of headache, lethargy, and fever (temperature of
103  F). In the 12 hours before her arrival, she had developed
nausea and vomiting. A computed tomography (CT) scan of
her head yielded normal findings. A complete blood count
identified mild neutrophilic leukocytosis and normocytic
anemia. A lumbar puncture was performed; the results
revealed leukocytosis with a predominance of neutrophils
(Image 1). Wright-Giemsa and Gram stains, performed on
slides and processed via a Cytospin instrument (Thermo
Fisher Scientific Inc), yielded positive results for numerous
organisms. Medical technologists in the hematology and
microbiology laboratories identified the organisms as
morphologically consistent with Naegleria species (Image 1
and Image 2) and informed the ED staff of their test result
interpretation 75 minutes after receiving the CSF specimen.
The preanalytic and analytic stages of CSF evaluation can
greatly affect diagnosis. At our institution, protocol dictates
www.labmedicine.com
Downloaded from https://academic.oup.com/labmed/article-abstract/47/2/149/2937887
by guest
on 25 August 2018
Case Study
that all fluids be sent to the laboratory for analysis
immediately and handled on a STAT (immediate) basis
because lysis of cells takes place soon after removal from
the body, generally within 1 hour of collection.12 The
specimen is evaluated grossly for color, turbidity, and the
appearance of the supernatant. Next, slides are prepared via
Cytospin centrifugation, and a cell count is performed on a
hemacytometer for white blood cells (WBCs) and red blood
cells (RBCs). The 2 Wright-Giemsa stained slides that had
been prepared via Cytospin are reviewed, and a medical
technologist performs a WBC differential count.
In the case of this patient, her CSF was described as milky in
color, with increased turbidity and a colorless supernatant.
The WBC count was 3675 per mL and the RBC count was 53
per mL; a manual differential of 100 cells identified 86%
neutrophils, 1% bands, 2% lymphocytes, 9% monocytes, and
2% other cells. Usually, a single Gram-stained slide is
prepared. However, due to the turbid nature of the fluid, the
elevated WBC count, and the increased neutrophil count, the
microbiology technologist (T.R.), concerned about the
possibility of meningitis, prepared 2 Gram-stained slides
from CSF specimens, processed them via Cytospin, and
examined them immediately.
Lab Medicine 2016;47:2;149–154 151
DOI: 10.1093/labmed/lmw008
 Case Study

Senior medical technologists in the hematology and

mircobiology laboratories identified the organisims as
morphologically consistent with Naegleria species

Medical technologists informed in ED staff

approximately 1 hour after receiving the CSF specimen

Infectious Disease was consulted immediately and the

patient was treated for PAM with amphotericin,
rifampin, azithromycin, fluconazole, and
dexamethasone (to control for cerebral edema)

CDC was contacted and Miltefosine, an investigational

medication, was started on day 2 of hospitalization

Amoeba culture on CSF identified amoebae, consistent

with Naegleria species

Real time PCR was Real time PCR was

performed at the CDC: performed at the CDC:
Positive for Naegleria Negative for
fowleri Acanthamoeba and
Balamuthia mandrillaris

Figure 1
Laboratory and clinical follow-up in treating the patient, a 12-year-old Caucasian girl. ED refers to emergency department; CSF, cerebrospinal
fluid; PAM, primary amoebic meningoencephalitis; CDC, Centers for Disease Control and Prevention; PCR, polymerase chain reaction.

In this case, the suspicious organisms were first identified in the
Gram-stained smear by a microbiology technologist. She
subsequently, the microbiology technologist evaluated the
Wright-Giemsa stained slides processed via Cytospin in the
Hematology division with 2 hematology technologists, including
the shift supervisor, and came to the conclusion of suspicious
diverse differential diagnosis. At the time of the laboratory
findings, no evidence existed in the clinical record that PAM
was a considered or suspected diagnosis at that time. On
the discovery of amoeba in the CSF, additional history
indicated the patient had been swimming at a freshwater
park 1 week before her arrival at the ER.

organisms resembling amoebae: likely, Naegleria species. The

Naegleria trophozoites are large, round to pear-shaped cells
with a prominent, dark nucleus and scattered vacuoles. On
Gram staining, the cells can appear similar to macrophages;
however, the cytoplasm of the amoebae has a well-defined
border. The diagnosis is noteworthy because the clinical
findings at the time of diagnosis included fever, headache, and
lethargy, all of these symptoms are nonspecific and offer a
Clinical Follow-Up
Immediately after the ED physicians received the CSF
results, they consulted the Division of Infectious Disease and
began to treat the patient for PAM with amphotericin,
rifampin, azithromycin, and fluconazole. She also received

152 Lab Medicine 2016;47:2;149–154 www.labmedicine.com

152 DOI: 10.1093/labmed/lmw008
Downloaded from https://academic.oup.com/labmed/article-abstract/47/2/149/2937887
by guest
on 25 August 2018

aggressive supportive therapy, including dexamethasone, to
control for cerebral edema. The ED physicians contacted the
Centers for Disease Control and Prevention (CDC) and on
day 2 of hospitalization, they started the patient on
miltefosine, an investigational medication, at 50 mg enterally
every 8 hours. Amoeba culture of CSF was performed by a
reference laboratory; the results identified the presence of
amoebae, consistent with Naegleria species. Culture results
were reported to the clinical team within 48 hours of the initial
arrival of the patient at the ED. Laboratory professionals at
the CDC performed real-time PCR on specimens from the
patient; the results were positive for N. fowleri (Figure 1). The
patient received 27 days of treatment with antifungals and
antibiotics, in addition to placement of an external ventricular
drain, hyperosmolar treatment with mannitol, and induced
hypothermia to lower intracranial pressure.9 The results of
repeat CSF studies during hospital days 10 through 16 all
had negative results for microorganisms. Further brain
imaging showed significant improvement, with resolution of
parenchymal edema.
The patient made a full recovery after 52 days of
hospitalization. At her 1-year follow-up visit, the patient was
functioning at baseline. Follow-up magnetic resonance
imaging (MRI) of the brain, performed 1 year after the patient
was fully cured of PAM, identified subtle gliosis of both
cerebral hemispheres involving the cortex and white matter.
Gliosis is a nonspecific finding of reactive changes to the
brain tissue. A subsequent MRI at the 18-month follow-up
visit revealed some encephalomalacia of the left frontal lobe
and cerebellum, with optic nerves having unremarkable
characteristics. Encephalomalacia is a softening or
degeneration of brain tissue; it is secondary to the
inflammatory immune response to the amoeba infection. We
observed no acute abnormalities via MRI.
Discussion
N. fowleri is a free-living, thermophilic amoeba commonly
found in soil and stagnant freshwater locations, especially in
the southern United States.15 A fatal case of PAM was
reported as far north as Minnesota,16 which highlights the
importance of clinical suspicion and history regardless of
geography. N. fowleri infection begins by the amoebae
entering the nasal cavity, usually through introduction of
water via submersion, diving, or splashing. After rapidly
www.labmedicine.com
Downloaded from https://academic.oup.com/labmed/article-abstract/47/2/149/2937887
by guest
on 25 August 2018
Case Study
tracking along the olfactory nerves and through the
cribriform plate, the amoebae are able to bind to extracellular
matrix glycoproteins on human host cells. Several
pathogenic mechanisms enable the amoeba to harm its host,
including secretion of pore-forming proteins, proteases, and
phospholipases, all of which likely contribute to
demyelination. This destruction leads to the lysis of
erythrocytes and surrounding nerve cells. The amoeba
also has immune-evasion mechanisms such as removal
of the membrane attack complex and resistance to
cytokines.4
PAM has a variable incubation time, ranging from 1 day to as
long as 7 days.17 The clinical signs have similarities with
those of bacterial and viral meningitis, including fever, neck
stiffness, and severe headaches.18 Symptoms can progress
to prolonged nausea, vomiting, and even seizures. The
disease can progress to acute hemorrhagic necrotizing
meningoencephalitis, which can lead to death in as soon as
7 to 10 days.4 CSF analysis reveals increased intracranial
pressure, an increase in neutrophils, and the presence of
amebic trophozoites consistent with N. fowleri. A variable
delay in treatment can be secondary to time intervals in
multiple stages of care, including exposure to exhibition of
symptoms; arrival for treatment at a health care facility; work-
up of the diagnosis (initial diagnosis of likely bacterial
meningitis);16 and finally, from diagnosis to initiation of
recommended therapy.
Published data have shown that as many as 40% of WBCs in
CSF may lyse after 2 hours at room temperature.19 Results of
another study12 verified that CSF has a decrease in
neutrophil counts at 1 hour by 32% and at 2 hours by 50%.
Other bodily fluids may show a similar decrease in cell
counts at room temperature. Therefore, our institutional
policy is that cell counts should be performed for all fluid
specimens within 2 hours of collection. If a delay is
anticipated, the specimen should be refrigerated. At our
institution, our goal is to interpret bodily fluid results
generally within 1 hour on all shifts. In the case we have
described herein, our hematology and microbiology
technologists identified an unusual organism, diagnosed it as
an amoeba (N. fowleri), and reported the result within 1 hour
and 15 minutes of the specimen being received in the
laboratory. The speed of the microscopic evaluation and
diagnosis played a major role in the ability of the clinical
teams to provide aggressive treatment and novel therapies
to the patient.
Lab Medicine 2016;47:2;149–154 153
DOI: 10.1093/labmed/lmw008
 Case Study
Pediatric practitioners, especially those who practice in the
southern United States during the summer months, should
be careful to include this differential in the setting of
meningitis-like symptoms.15 Further, this diagnosis should
be considered by medical technologists when evaluating a
CSF specimen that has indicators of meningitis, including
turbid fluid, elevated WBC count, and increased neutrophil
count. Successful treatment of PAM is a rare occurrence and
can only be attempted after correct diagnosis, which relies
on rapid recognition of the microorganism by medical
technologists and pathologists. In our patient, prompt
diagnosis, followed by rapid communication with the treating
physicians in the ED and consultation with Infectious
Disease specialists, led to the patient being fully cured of
PAM. Therefore, it is critical that medical technologists
consistently provide timely CSF evaluation, explore the
diagnosis of PAM, and look for amoebae in the setting of
meningitis, especially in the summertime. LM
References
1. De Jonckheere JF.What do we know by now about the genus Naegleria?
Exp Parasitol. 2014;145(Suppl):S2–S9.
2. De Jonckheere JF.Origin and evolution of the worldwide distributed
pathogenic amoebaflagellate Naegleria fowleri. Infect Genet Evol.
2011;11(7):1520–1528.
3. Heggie TW. Swimming with death: Naegleria fowleri infections in recre-
ational waters. Travel Med Infect Dis. 2010;8(4):201–206.
4. Marciano-Cabral F, Cabral GA. The immune response to Naegleria fowleri
amebae and pathogenesis of infection. FEMS Immunol Med Microbiol.
2007;51:243–259.
5. Grace E, Asbill S, Virga K. Naegleria fowleri: pathogenesis, diagnosis, and
treatment options. Antimicrob Agents Chemother.
2015;59(11):6677–6681.
6. Rojas-Herna ndez S, Jarillo-Luna A, Rodriguez-Monroy M, Moreno-
Fierros L, Campos-Rodriguez R. Immunohistochemical characterization
154 Lab Medicine 2016;47:2;149–154
154 DOI: 10.1093/labmed/lmw008
Downloaded from https://academic.oup.com/labmed/article-abstract/47/2/149/2937887
by guest
on 25 August 2018
of the initial stages of Naegleria fowleri meningoencephalitis in mice.
Parasitol Res. 2004;94(1):31–36.
7. Seidel JS, Harmatz P, Visvesvara GS, Cohen A, Edwards J, Turner J.
Successful treatment of primary amebic meningocephalitis. N Engl J
Med. 1982;306(6):346–348.
8. Vargas-Zepeda J, Go mez-Alcala  AV, V
asquez-Morales JA, Licea-Amaya
L, De Jonckheere JF, Lares-Villa F. Successful treatment of Naegleria
fowleri meningoencephalitis by using intravenous amphotericin B, fluoco-
nazole and rifampicin. Arch Med Res. 2005;36(1):83–86.
9. Linam WM, Ahmed M, Cope JR, et al. Successful treatment of an adoles-
cent with Naegleria fowleri primary amebic meningoencephalitis.
Pediatrics. 2015;135(3):e744–e748.
10. Visvesvara GS, Moura H, Schuster FL. Pathogenic and opportunistic
free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris,
Naegleria fowleri, and Sappinia diploidea. FEMS Immunol Med Microbiol.
2007;50:1–26.
11. Martınez AJ. Free-living amebas: natural history, prevention, diagnosis,
pathology, and treatment of disease. CRC Press. 1985;1–190.
12. Steele RW, Marmer DJ, O’Brien MD, Tyson ST, Steele CR. Leukocyte
survival in cerebrospinal fluid. J Clin Microbiol.1986;23(5):965–966.
13. Deisenhammer A, Baros A, Egg R, Gilhus NE, Giovannoni G,
Rauer S, Sellebjerg F. Guidelines on routine cerebrospinal fluid
analysis. Report from an EFNS task force. Eur J Neurol.
2006;13:913–922.
14. Fleming C, Russcher H, Lindemans J, de Jonge R. Clinical relevance
and contemporary methods for counting blood cells in body fluid
suspected of inflammatory disease. Clin Chem Lab Med.
2015;53:1689–1706.
15. Lopez C, Budge P, Chen J, et al. Primary amebic meningoencephalitis: a
case report and literature review. Pediatr Emerg Care.
2012;28(3):272–276.
16. Kemble SK, Lynfield R, DeVries AS, et al. Fatal Naegleria fowleri infection
acquired in Minnesota: possible expanded range of a deadly thermophilic
organism. Clin Infect Dis. 2012;54(6):805–809.
17. Yoder JS, Eddy BA, Visvesvara GS, Capewell L, Beach MJ. The epidemi-
ology of primary amoebic meningoencephalitis in the USA, 1962-2008.
Epidemiol Infect. 2010;138(7):968–975.
18. Abrahams-Sandı E, Retana-Moreira L, Castro-Castillo A, Reyes-Batlle M,
Lorenzo-Morales J. Fatal meningoencephalitis in child and isolation of
Naegleria fowleri from hot springs in Costa Rica. Emerg Infect Dis.
2015;21(2):382–384.
€hrborn A, Annas M, Faustmann P, Lennartz K,
19. Dux R, Kindler-Ro
Zimmerman CW. A standardized protocol for flow cytometric ana-
lysis of cells isolated from cerebrospinal fluid. J Neurol Sci.
1994;121:74–78.
www.labmedicine.com