Case Study. Andrew L. Dunn, MD, 1 Tameika Reed, MT (ASCP), 2 Charlotte Stewart, MT (ASCP), 2 Rebecca A. Levy, MD 1,2 * ABSTRACT

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1 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, MD 1,2 * Laboratory Medicine 47:2: DOI: /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 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 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 system; CSF, cerebrospinal fluid; PCR, polymerase chain reaction; ED, emergency 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, 2 Department of Pathology, Clinical Laboratory Arkansas Children s Hospital, Little Rock *To whom correspondence should be addressed. RALevy@uams.edu 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 welldocumented 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 VC American Society for Clinical Pathology, All rights reserved. For permissions, please journals.permissions@oup.com 149

Image 1 Naegleria fowleri on Wright-Giemsa stained cerebrospinal fluid cytospin slides (original magnification, 1000; oil immersion). Arrows indicate Naegleria organisms.

2 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 studies 12,13 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; DOI: /labmed/lmw008

Image 2 Naegleria fowleri on Gram-stained cerebrospinal fluid cytospin slides (original magnification, 500; oil immersion). Arrows indicate Naegleria organisms.

3 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 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; DOI: /labmed/lmw008

4 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 performed at the CDC: Positive for Naegleria fowleri Real time PCR was performed at the CDC: Negative for 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 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 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. 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; DOI: /labmed/lmw008

5 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 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; workup 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 study 12 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; DOI: /labmed/lmw008

6 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): Heggie TW. Swimming with death: Naegleria fowleri infections in recreational waters. Travel Med Infect Dis. 2010;8(4): Marciano-Cabral F, Cabral GA. The immune response to Naegleria fowleri amebae and pathogenesis of infection. FEMS Immunol Med Microbiol. 2007;51: Grace E, Asbill S, Virga K. Naegleria fowleri: pathogenesis, diagnosis, and treatment options. Antimicrob Agents Chemother. 2015;59(11): Rojas-Hernandez S, Jarillo-Luna A, Rodriguez-Monroy M, Moreno- Fierros L, Campos-Rodriguez R. Immunohistochemical characterization of the initial stages of Naegleria fowleri meningoencephalitis in mice. Parasitol Res. 2004;94(1): 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): Vargas-Zepeda J, Gomez-Alcala AV, Vasquez-Morales JA, Licea-Amaya L, De Jonckheere JF, Lares-Villa F. Successful treatment of Naegleria fowleri meningoencephalitis by using intravenous amphotericin B, fluoconazole and rifampicin. Arch Med Res. 2005;36(1): Linam WM, Ahmed M, Cope JR, et al. Successful treatment of an adolescent with Naegleria fowleri primary amebic meningoencephalitis. Pediatrics. 2015;135(3):e744 e 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: Martınez AJ. Free-living amebas: natural history, prevention, diagnosis, pathology, and treatment of disease. CRC Press. 1985; Steele RW, Marmer DJ, O Brien MD, Tyson ST, Steele CR. Leukocyte survival in cerebrospinal fluid. J Clin Microbiol.1986;23(5): 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: 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: Lopez C, Budge P, Chen J, et al. Primary amebic meningoencephalitis: a case report and literature review. Pediatr Emerg Care. 2012;28(3): 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): Yoder JS, Eddy BA, Visvesvara GS, Capewell L, Beach MJ. The epidemiology of primary amoebic meningoencephalitis in the USA, Epidemiol Infect. 2010;138(7): 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): Dux R, Kindler-R ohrborn A, Annas M, Faustmann P, Lennartz K, Zimmerman CW. A standardized protocol for flow cytometric analysis of cells isolated from cerebrospinal fluid. J Neurol Sci. 1994;121: Lab Medicine 2016;47:2; DOI: /labmed/lmw008

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