Clinical Infectious Diseases Advance Access published August 22, 2012

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Primary Amebic Deaths Associated With Sinus Irrigation Using Contaminated Tap Water

Jonathan S, Yoder, 1 Susanne Straif~Bourgeois, 4 Sharon L. Roy, 1 Thomas A. Moore,5 Govinda S. Visvesvara, 1 Raoult C. Ratard, 4 Vincent R. Hill.1 Jon D. Wilson,6 Andrea J. Linscott,5 Ron Grager,7 Natalia A. Kozak,2 Rama Sriram,1 Jothikumar Narayanan,1 Bonnie Mull,1 Amy M. Kahler, 1 Chandra Schneeberger,1 Alexandre J. da Silva,3 Mahendra Poudel,5 Katherine L Baumgarten,5 Lihua Xiao.1 and Michael J. Beach1 1National Center for Emerging and Zoonotic Infectious Diseases, 2National Center far Immunization and Respiratory Diseases, and 3Center for Global Health, Centers for Disease Control and Prevention, Atlanta, Georgia; 4Louisiana DEpartment of Health and Hospitals, and 50chsner Health System, New Orleans, 60epartment of Pathology, Louisiana State University Health Science Center-Shreveport and 7DeSoto Parish Coroner, Mansfield, Louisiana Background. fowleri is a climate-sensitive, thermophilic ameba found in the environ1nent, including warm, freshwater lakes and rivers. Primary amebic meningoencephalitis (PAM), which is almost universally fatal, occurs when N. fowleri-containing water enters the nose, typically during swllnming, and N. fowleri migrates to the brain via the olfactory nerve. In 2011, 2 adults died in Louisiana hospitals of infectious meningoencephalitis after brief illnesses. Methods. Clinical and environmental testing and case investigations were initiated to determine the cause of death and to identify the exposures. Results. Both patients had diagnoses of PAM. Their only reported water exposures were tap water used for household activities, including regular sinus irrigation with neti pots. Water samples, tap swab san1ples, and neti pots were collected fron1 both households and tested; N. fowleri were identified in water samples fro1n both homes, Conclusions. These are the first reported PAM cases in the United States associated with the presence of N. fowleri in household plwnbing served by treated municipal water supplies and the first reports of PAM potentially associated with the use of a nasal irrigation device, These cases occurred in the context of an expanding geograph­ ic range for PAM beyond southern tier states with recent case reports fro1n Minnesota, Kansas, and Virginia. These infections introduce an additional consideration for physicians recommending nasal irrigation and demon­ strate the importance of using appropriate water (distilled, boiled, filtered) for nasal irrigation. Furthennore, the changing epide1niology of PAM highlights the importance of raising awareness about this disease a1nong physi­ cians treating persons showing n1eningitislike symptoms.

Many species of Naegleria are found in the environ- thern1ophilic, free-living aineba that is frequently de­ 1nent [1] but only l species, Naegleria fowleri, causes tected in natural bodies of wann freshwater, where it a rare but serious disease called primary arnebic feeds on bacteria. It tolerates te1nperatures of up to 45° rneningoencephalitis (PAM) [2-4]. N. fowleri is a C and thrives during wanner months of the year when the ambient temperature increases. This organisn1 has been isolated from freshwater lakes, ponds, and rivers Roceived 20 April 2012; acceptod 19 June 2012. [5-7], hot springs [8], thermally polluted water [9-llj, Corresprmdence: Jonathan S. Yoder, MSW, MPH, National Center for Emerging \.vann groundwater [12], inadequately treated swiln­ and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd. MS C"08, Atlanta. GA 30329 ([email protected]), n1ing pools [13] , sewage [14, 15] and soil [16, 17}. Clinical lnfElctious Diseases PM1 typically occurs after exposure to warn1, untreat­ Published by Oxford University Pross on behalf of the Infectious Diseases Society of ed water, frequently associated with swiin1ning. The America 2012. DOI: 1Q_1093/cid/cis626 n1edian age of case patients in the United States is 12

Deaths Caused by Naegleria fowler! Infections • CID • 1 years> with the majority of infections occurring in males [3]. patient was urgently transferred to a neurologic critical care N. fowleri enters the nose, and migrates along the olfactory unit. Mannitol and dexamethasone were used to decrease in­ nerve through the cribriforn1 plate to the brain. Onset of syinp­ tracranial pressure. Overnight, he remained intubated and hy­ toms occurs 1-7 days after exposure [18]. Signs and symptoms potensive, requiring vasopressor 1nedications to stabUize his of infection are similar to those of bacterial or viral blood pressure. Serial neurologic examinations demonstrated and include headache, fever, stiff neck, anorexia, vomiting, rapid decline in response to verbal commands followed by altered mental status, seizures, and coma. The 1nedian time fixed, n1idrange pupils. On 8 June, con1puted tomography from symptom onset to death typically is 5 days [18]. with contrast enhancement showed con1plete sulcal effacement and diminished ventricle size consistent with increased intra­ cranial pressure. The patient's neurologic condition re111ained CASE REPORTS unchanged, and he was declared brain dead. On 10 June, real­ time PCR testing at the CDC confirmed the diagnosis of PAM Case 1 due to N. fowleri; N. fowleri were also cultured from the CSF On 5 June 2011, a 28 year-old n1an from southern Louisiana (Table 1). An1plification and bidirectional sequencing of the abruptly developed a severe occipital headache radiating down intern.al transcribed spacer and mitochondrial small subunit his neck, neck stiffness, back pain, and intermittent vomiting. ribosomal Rl'\/A genes [20] identified genotype I. :E-Ie had a history of n1igraines and thus did not seek medical On 17 June 2011, the Louisiana Department of Health and attention at symptom onset. On 6 June he was brought to a Hospitals began an epidemiologi.c investigation of the first New Orleans hospital einergency department with confusion. case. The patient's mother, with whom he liVed, and his co­ On initial examination, the patient was febrile (temperature, workers lmew of no recent history of recreational freshwater 38.5°C), disoriented, and combative. Further examination re·· contact ( eg, fishing, boating, swimming, diving, or tubing). vealed meningismus but no gross rnotor deficits or cranial The patient had chronic allergic sinusitis and irrigated his nerve abnormalities. No skin lesions were noted. Pending a sinuses with a neti pot (Figure 1) at least once daily, using tap lumbar puncture, glucocorticoids and empiric anti-infective water to which he added a commercially available salt packet therapy (intravenous ceftriaxone, linezolid, and acyclovir) (same brand as the irrigation device)< For convenience, he were started. The patient was emergently intubated and kept the device next to the bathroo1n sink. sedated for airway protection. Water samples from the nlunicipal water treatment plant On admission, peripheral blood examination showed an el­ and distributiort system, water samples and swabs frorn the evated white blood cell count. Rapid human in1n1unodeficien­ patient's household, and the irrigation device were sent to the cy virus test results were negative. Noncontrast head CDC for further testing (Table 1). Water temperature and computed ton1ographic findings were normal. Lu1nbar punc­ total chlorine residual 1neasurements were made on some of ture revealed grossly turbid cerebrospinal fluid (CSF); opening the water samples at the time of collection. Water ten1pera­ pressure was not measured but appeared to be elevated t.ures in the prernise plumbing hot water systen1 (a tankless because CSF projected 6 cm out of the needle on removal of water heater) ranged from 103'F to 113'F (39'C-45'C). the stylet. CSF results suggested acute bacterial meningitis, al­ No amebae were detected in the samples from the nlunici­ though the red blood cell count was elevated (343 cells/mL), pal water treatlnent plant and distribution system serving this and the white blood cell differential showed that 17% were household (Table 1). However, multiple types of an1ebae "other" cells. The CSF Gram stain showed 1nany neutrophils (Hartmannella, Vannella, and Naegleria sp.) were detected in but no 1nicroorganis1ns. The negative Gran1 stain prompted a some of the san1ples taken at various locations within the wet 1nount by laboratory personnel the next n1orning, 7 June household. Water collected. fron1 the tankless water heater was which clearly identified motile ameboid trophozoites. Further culture positive for Naegleria sp. and Hartmannella sp. and review of the CSF cytospin or "cytocentrifuge" preparation was PCR positive for N. jowleri; the genotype could not be fro1n the night before revealed that the "other" cells were identified. The sa1nple was also culture positive for Legionella an1eboid trophozoites. Ilnages were sent to the Centers for pneumophila. The neti pot was culture positive for Hartnian­ Disease Control and Prevention (CDC) DPDx Web site (http:// nella sp. but PCR negative for N. fowleri. www.dpcl.cdc.gov/dpdx/Default.ht1n) for telediagnosis that af­ The household occupants were advised to remediate the ternoon and the presumptive diagnosis of amebic 111eningoen­ pre1nise plumbing by setting the tankless 1-vater heater thern10- cephalitis was supported, pending testing by real-tin1e stat at a level at which each of the distal taps would have a polymerase chain reaction (PCR) [19] and culture. water ten1perature of 160°F (71°C) and to initially run all taps Treat111ent with liposornal and rifa1npin (one faucet at a tin1e) for at least 5 1ninutes to kill any remain­ began abnost iln1nediately after trophozoite identification. The ing N. fowleri (and Legionella) in the water heater and prernise

2 • CID • Yoder et al 'f111Jl!M-et iniC11!•Sput:imeiranit'Environmenta 1-SampTir-Test"RffSulti>f or-C'!lsl!l!"!lf"'PrimarrAm &b lt:""Metii ng oem:'epha Iitis:-toil i•i•mr.-~ 2011

Naegleria fowleri PCR 8 Total Chlorine Culture (or IF Al Result Result Temperature °F {°C) Residual, mg/Lb

Sample Type and Source Patient 1 Patient 2 Patient 1 Patient 2 Patient 1 Patient 2 Patient 1 Patient 2 Clinical Patient CSF N. fowleri NT Positive, NT NA NA NA NA genotype ·1 Autopsy specimen (brain NT N. fowleri (by NT PoSitive NA NA ·NA NA tissue) IFAI Water treatment plant Reservoir Negative NT Negative Negative 77 (25) 51 110) 2.99 3.9 Clear well Negative NT Negatve 77 (25) NT 3.83 NT tJ NT 0 Distribution system Negative i Tower NT Negatve Negative NT 53 111) 0.2 I.I p. "0 Water main s.erving Negative NT Negatve NT NT NT 0.16 NT p. case patient's 3' neighborhood 8 Point of entry of Negative NT Negative Negative NT 99 (37) 0.22 0.61 :g municipal water into ::::: 0. case patient's residence ""0x 8' Hous6hold ~ & Swab samples § 3 Bathroom tap Negative Negative Negative NT NT NT NT NT • Main shower nozzle Hartmannel!a Negative Negative NT NT NT NT NT "0 ~ Bathtub -faucet NT Hartmannella NT NT NT NT NT NT 0• Toilet tank sediment Hartmannef/a Negative Negative NT NT NT NT NT tJ Handheld shower Vanne!fa NT Negative NT NT NT NT NT 0 nozzle ""§:: Kitchen faucet Negative Hartmanneffa Negative NT NT NT NT NT 0 kitchen spray nozzle HartmBnnefla NT -Negative NT NT NT NT NT "0 Dishwasher nozzle Negative Negative Negative NT NT NT NT NT "So t"' Dishwasher dr8in Negative Negative Negative NT NT NT NT NT O' Water samples ~ R> Haridheld shower Negative NT Negative NT 110 (43.3) NT NT NT s< nozzle 8' Kitchen faucet Negative Amebaec Negative Positive 112 144.5) 91 132.5) NT 0 3 Main shower nozzle Haitmannelfa Amebaec Neg9tive Positive 103 (39.3) 115 (46.1) NT 0.02 •5· "0 Bathtub faucet NT N. fowler! NT Positive, NT 115 (46.3) NT 0.02 0 genotype 1 ~ Bathroom sink faucet Negative N. fowler! Negative Positive, 107 (41.5) 99 (37.0) NT 0 0 genotype 1 ")> Water heater Hartmannel/a, Negative Positive Negative 113 (448) NT NT 0.09 " Naegferia ~ N sp.d -~ N Neti pot Hartmanne/!a Negative Negative NT NT NT NT NT s 'v Abbreviations: CSF, cerebrospinal fluid; IFA, immunofluorescence assay; NA, not available: NT, not tested: PCR, polymerase chain reaction. " Real-time PCR analysis was used for det0ction of N. fowler!. Molecular characterization was attempted on available samples through amplification and bidirectional sequencing of the internal transcribed spacer and mitochondrial small subunit ribosomal RNA genes; where available, genotype information is provided. b The municipal water systems used monochlorarnine for residual disinfection. The expected range for total chlorine residual in distribution systems is 0.2-4 mg/L. c Amebae observed but not characteristic of N. fowleri. d The water sample from the tankless water heater for patient 1 was also positive by culture for non-serogroup 1 Legione/la pneumophila.

Deaths Caused by Naegleria fowleri Infections • CID • 3 and total chlorine residual measurement's were ta::tten on water ft san1ples at the time of collection. Water temperatures in the system, including the water heatel' tank, ranged fro1n 91°F to llS'F (33'C-46'C). Water samples collected from the kitchen faucet, shower, bathtub faucet, and bathroom sink faucet tested positive by direct PCR for N. fowleri; further identified as genotype 1. No an1ebae were cultured fro1n the neti pot. The ability to recover organisrr1s fron1 the neti pot inight have been di1ninished by the 2-month delay between the last usage of the device and testing; furthermore, the neti pot was dry when tested. The fan1ily was advised to re1nediate the hot water system, as directed for the first case.

Figure 1, Neti pot. N. fowleri Inactivation Testing Using Neti Pot Saline Solutions The ability of comrr1ercially available reconstituted salt packets in the neti pot to inactivate N. fowleri was tested at the CDC plumbing, subsequently lowering the thermostat to about 120° laboratory. Saline solution packets fro1n 2 companies were pre­ F to reduce the risk of scalding. To reduce the risk of recoloni­ pared according to the manufacturers' directions. A saline sol­ zation, the occupants were further advised to repeat this pro­ ution packet was dissolved in 240 mL of distilled water to cedure every few weeks. achieve a solution with 0.9% sodiun1 chloride. Experiments were set up in duplicates in Costar 24-well Cluster flat-bottom Case 2 plates with lids. N. fowleri amebae from the first case patient On 28 September 2011, a 51-year-old won1an from northern actively growing in modified Nelson mediu1n were adjusted to Louisiana was admitted to the hospital with a 3-day histo1y of obtain 500 amebae per milliliter of the saline solutions, and 2 altered mental status, nausea, vomiting, poor appetite, listless­ n1L of this solution was dispensed into each well and incubat­ ness, fatigue, and high fever. On examination, she was febrile ed in moist cha1nbers at 37°C for 1, 4, and 18 hours; 2 mL of (temperature, 38.6°C) and lethargic with neck stiffness and N. fowleri an1ebae (500 amebae per n1i1liliter) in growth thyromegaly. Meningitis was suspected based on con1plete mediu1n were set up as controls. At the end of each time blood cell count, CSF findings, and physical examination, al­ period the amebae in the wells were photographed with an though tests for specific viral, bacterial, and fungal Olympus IMT-2 inverted rnicroscope equipped with a digital were negative, as were bacterial culture. No gross n1otor defi­ camera. Test results revealed that the nu1nber of N. fowleri or­ cits or cranial nerve abnormalities were found. The patient ganisms did not appreciably decrease or degrade after 4 hours; died on 2 October. An autopsy was perfonned by the Foren­ observation at 18 hours revealed that inore than half of the sics Unit at the Louisiana State University Health Science an1ebae had died (cells rounding up or lifting off plate) Center-Shreveport Departrnent of Pathology, and a1nebic Inf> (Figure 2). The experiment was replicated with N. fowleri ningoencephalitis was identified. Based on the distribution of amebae isolated fro1n 2 unrelated fatal infections, with similar gross alterations affecting the brain, and absence of obvious results. cyst forms, Naegleria sp. was suspected. On 18 November, brain tissue forwarded to the CDC for confinnatory testing DISCUSSION tested p~itive for N. fowleri by_2._!n1nunohistochemistry, prompting an epiden1iologic investigation. The genotype PAM cases and deaths associated with exposure to tap water could not be identified. within a household highlight the changing epidemiology of The patient lived by herself; her parents lived on the same N. fowleri in the United States. Previously, cases in the United street block. According to her parents, the patient did not States usually occurred ainong persons recreating in wann have any recreational freshwater exposure in the 2 weeks freshwater in southen1-tier states [18}. The 2 cases in Louisiana before illness onset. Because the patient had sinus proble1ns, in 2011 represent the first tllne disinfected tap water has been she regularly used a neti pot, especially after 1-vorking in the in1plicated in N. fowleri infection. Although N. fowleri are ubiq· yard, to ren1ove dust from her nasal area. Her parents doubted uitous in freshwater aquatic environ1nents, tap water in the that she used distilled, filtered, or previously boiled water for United States has previously been ilnplicated only as an uncon1- nasal irrigation, Water san1ples and swab sa111ples fro1n the n1on exposure source. Two PAM cases in Arizona (2002) were patient's residence were collected (Table 1). Water te1nperature associated with untreated geothennal inunicipal well water [21]

4 • CID • Yoder et al 1 hour

4 hours

18 hours

N. fowler/in c;omme-rcially a\/all~ble reconstituted saline N, fowleri in growth medium (control) packets intended for nasal irrigation

Figure 2. Naeglerfa fowferi exposure to a commercial saline solution designed for nasal irrigation, by length of exposure (left) and N, fowler! in growth medium (control; right; viewed at x600 magnification). After 1 hour, there was no decrease or increase in numbers of amebae in saline or growth medium: after 4 hours, there was no decrease or increase in the number ot amebae in saline solution but a twofold increase in numbers in growth medium; after 18 hours, more than half of the amebae were dead in saline solution and good growth continued in growth medium.

[When the cases occurred, there was no treatlnent of the mL1- an1plify in warm water niches such as water heaters, and nicipal water supply by inethods of chlorination, ozonation, UV shower heads [26, 27]. Naegleria spp. also seem to be able to irradiation, or filtration (20)]. These Louisiana cases represent colonize biofilm.s and have been isolated from premise plun1b­ the first reported cases in the United States associated with ing served by both treated and untreated inunicipal water sup­ treated municipal water, although other such cases have been plies [21, 28]. N. fowleri cysts and trophozoites are fairly reported in Australia [22] and Pakistan [23]. The municipal resistant to chlorine disinfection [29, 30]. Although N. fowleri wa!E_r distribution systeins for .!?..0l1 Louisiana cases was chlora- were no!.. isolated fro1n the n1unicipa~.-water system in the Lou­ 1ninated and total chlorine residual levels ranged from 0.2 to isiana cases, Naegleria spp. were isolated fro1n the pre1nise 1.1 n1g/L in the distribution syste1ns (Table 1), thereby meeting plumbing, along with other a1nebae. It is unclear how US Environmental Protection Agency (EPA) regulations for N. fowleri were introduced into the premise plu1nbing of these treated drinking water fro1n surface water supplies [24] (N. patients' houses. I-Iowever, once introduced, they were able fowleri is not an1ong the >90 contan1inants under national to colonize the hot water syste1ns of these hon1es. Because drinking water regulation by the EPA but has been on EPA's these were the first US PAM cases associated with residential Contaminant Candidate List 3 [25]). disinfected drinking water supplies, no forn1alized N. fowleri Environmental pathogens (eg, Pseudomonas aeruginosa, Le­ remediation and prevention guidelines exist for this setting. gionella spp., nontuberculous n1ycobacteria) have ernerged as Temperatures used for Legionella rernediation in nonre.siden­ well-docun1ented biofiln1 colonizers within pipes found in tial building systen1s and centralized syste1ns in multifamily 1nanmade, engineered environn1ental habitats where they residential buildings should also kill N. fowleri and forn1ed the

Deaths Caused by Naegleria fowleri Infections • CID • 5 Naeglerfa fowler! infects people by entering the body through contam_inatidn of tap water and the ability of N. fowleri to the nose, A person cannot be infected with N. fowleri by survive for short times in nasal irrigation salt solutions made dl'inking contaminated water. Personal actions that might with conta1ninated tap water. As a result, these persons should reduce the risk of infection Include: ensure that water used to create nasal irrigation solutions is Nasal rinses or sinus irrigation: distilled, filtered (using a filter with an absolute pore size of '.'.! µrn ), or previously boiled (Figure 3). Do not use tap water or untreated freshwater, When Recently, the geographic pattern of this climate-sensitive, Irrigating, flushing, or rinsing nasal passages, use st9rl!e, thermophilic- ameba seems to be changing, with single cases distilled, flltered (u$1ng s filter with an absolute pore size of 1 µm or sma!ler) or previouslybotledwaterto make the recently reported for the fir?t time in Minnesota (2010) and 1 irrigation solution. Kansas (2011), and for the first tin1e since 1969 in Virginia ·---·Rfnse the 1rrigat1o·n~aeV1ce aft~r· e'a~h ·use us1ngTh_e_sa_m_e---~c2011) [18, 34JlCDCUTIPUbliST1ed"data)~fheSe"'C8.Se"SOCClii'fed'"~---- sterile, d!st!lled1 filtered, or previously boiled water, in warm freshwater locations after localized heat waves and might reflect ·an expansion of the geographic range of PAM or Recreationatwater activities: increased N. fowleri activity in northern clin1ates. It is unclear Avoid getting water up the nose, Hold the nose shut or use whether the increased te1nperature and heat waves projected nose dips when taking part in water-related activlties in climate change models will lead to further expansion of the involving warm freshwater. geographic range. To better address this, improved and sys­ Avoid digging In 9r stirring up the sediment while taking part tematic environmental san1pling of water bodies and systems In water-relatedactlvJtles Jn warm freshwater bodies, Avoid water-related actlv!t!es tn warm freshwater during in the United States is needed to create a baseline for N. perlods of high water temperature, fowleri occurrence, followed by systematic monitoring over time to bet'ter interpret, ·understand, and predict potential Figure 3. Measures to reduce the risk for primary amebic meningoen­ changes in N. fowleri ecology that could lead to improved pre­ cephalitis due to Naeg!eria fowleri. vention activities. In addition, developing efficacious therapies and raising the level of clinical awareness about PAM infec- @.. tions may improve the prognosis for future patients.

basis for the household pre1nise plumbing re1nediation recon1- Notes

1nendations in these cases [30, 31]. Preremediation water ten1- Acknowledgments. The following persons assistt:U. wilh Lh~ diuical, peratures measured in these households were far below these epidemiological, and laboratory portions of this investfgation: Mahendra Poudel, MD, Asher Shahzad, MD, Katherine L. Baun1ga1ten, MD, Lourdes levels. Re1nediation recon1mendations were specific to these Lago, MD, Vivek Sabharwal, MD, Erik T. Sundell, MD, Ochsner Health households and situations and should not be seen as general Systems; Nancy Hartwell, Louisiana State University Health Sciences recommendations for all homeowners. No data are available Center, Shrevep01t; Gary Balsan10, DVM, Keasha Henson, MPH, Louisiana on the prevalence of N. fowleri in other residential units Department of Health and Hospitals; Ellen Brown, AS, Lauri Hicks, DO, Claressa Lucas, PhD, Jonas Winchell, PhD, National Center for Im1nuniza­ served by the same water utilities or in other Louisiana and tion and Respiratory Diseases; Dawn M. Roellig, l\IIS, PhD, Rebecca Bandea, US communities. National Center for Emerging and Zoonotic Infectious Diseases, CDC. Nasal irrigation using saline solutions has been advocated Financial support. The authors con1pleted this work in the course of their work for their affiliated institutions and received no additional as a safe, inexpensive n1ethod for managing chronic allergic funding. rhinosinusitis [32]. However, the PAM cases reported here Potential conflicts of interest. All authors: No reported conflicts. and others in Australia [22] and Pakistan [23] involving direct All authors have subn1itled the ICMJE Fonn for Disclosure of Potential Conflicts of Interest Conflicts that the editors consider relevant to the or forceful application of tap water into the nasal passages (eg, content of the manuscript have been disclosed. directing- shower water up the nose,l·eligious ablutions) indi­ cate that there is a small risk associated with this practice References when tap water is used. Although N. jowleri cannot survive in 1. De Jonckheere JF. Molecular definition and the ubiquity of species in the level of salinity found in 111arine enviro111nents [33], the genus Naegleria. Protist 2004; 155:89-103. adding salt mixtures to tap water to prepare and rapidly use 2. Visvesvara GS, Moura H, Schuster FL. Pathogenic and opportunistic free-living an1oebae: spp., Balamulhia mandrillaris, nasal irrigation solutions does not seen1 to inactivate N. Naegleria fowleri, and diploidea. FEMS I1nn1lm Med Micro­ fowleri fast enough. The length of contact tin1e found in real bial 2007; 50:1-26. world conditions ( <1 n1inute) would probably not effectively 3. Marciano-Cabral F, Cabral GA. The irnnnu1e response to Naeg/eria inactivate N. fowleri, which probably requires hours for full Jowleri ai11ebae and pathogenesis of infection. FEMS Immun lv[ed lvli­ crobiol 2007; 51:243-59. inactivation (Figure 2). Persons practicing nasal irrigation 4. Visvesvara GS, Roy SL, Maguire JI-L Pathogenic and opportunistic should be aware of the likely s1nall risk for lY. fowleri free-living an1ebae: Acanthamoeba spp., ,

6 • CID • Yoder et al Naegleria fowleri, and Sappinia pedata, In: Guerrant RL, Walker DH, Balamutl1ia mandril/aris, and Naegleria fowleri. J Clin Microbial Weller PF, eds. Tropical infectious diseases-principles, pathogens 2006; 44:3589-95. and practice. 3rd ed. Edinburgh, UK: Saunders Elsevier, 2011: 20. Zhou L, Sriram R, Visvesvara GS, Xiao L. Genetic variations in the '707--13, internal transcribed spacer and n1itochondrial small subunit rRNA 5, Wellings FM, Amuso PT, Chang SL, Lewis AL. Isolation and identifi­ gene of Naeglerla spp. J Eukaryot Microbial 2003; 50(Suppl):522-6. cation of pathogenic Naegleria from Florida lakes, Appl Environ Mi­ 21. Marciano-Cabral F, MacLean R, Mensah A, LaPat-Polasko L. Identifi­ crobiol 1977; 34:661-7. cation of Naegleria fowleri in domestic water sources by nested PCR. 6. John DT, Howard MJ. Seasonal distribution of pathogenic free-living Appl Environ Microbiol 2003; 69:5864-9. an1ebae in Oklahoma waters. Parasite! Res 1995; 81:193-201. 22, Dorsch MM, Cameron AS, Robinson BS. The epidemiology and 7. Ettinger IvIR, Webb SR, Harris SA, Mdninch SP, Garman GC, Brown control of primary amoebic 1neningoencephalitis with particular refer­ BL. Distribution of free-living an1oebae in Jan1es River, Virginia, USA. ence to South Australia, Trans R Soc Trap Med Hyg 1983; 77:372-7. Parasitol Res 2003; 89:6-15. 23. Shakoor S, Beg MA, Mah1nood SF, et al, Primary amebic meningoen­ 8. Sheehan KB, Fagg JA, Ferris MT, Henson JivI. PCR detection and anal­ cephalit!s caused by Naegleria fowleri, Karachi, Pakistan, Emerg Infect ysis of the free-living an1oeba Naegleria in hot springs in Yellowstone Dis 2011; 17:258-61. and Grand Teton National Parks. Appl Environ Microbial 2003; 24. United States Environmental Protection Agency. Comprehensive 69,5914-8. surface water treatn1ent rules quick reference guide: systems using con­ 9. De Jonckheere J, Van Dijck P, Van de Voorde H. The effect of ventional or direct filtration 2010, Report 816-F-10-074. Available at: thermal pollution on the distribution of Naegleria fowleri. J Hygiene http ://www.epa.gov I ogwd w/ m db p/ pd fs/ qrg_n1d bp _sur facewatertrea t 1975; 75:7-13. rnent_convent_direct.pdf. Accessed 24 July 2012, 10. Behets J, Dederck P, Delaedt Y, Verelst L, Ollevier F. Survey for the 25. United States Environn1ental Protection Agency. Contaminant candi­ presence of specific free-living amoebae in cooling waters fron1 date list 3 - CCL. Available at: http://water.epa.gov/scitech/d.rinking Belgian power plants, Parasitol Res 2007; 100:1249-56. water/dws/ccl/ccl3.cfm. Accessed 24 July 2012. 11. Tyndall RL, Ironside KS, Metler PL, Tan EL, Hazen TC, Fliern1ans 26. Falldnham JO III. Mycobacterial aerosols and respiratory disease. CB. Effect of thermal additions on the density and distribution of En1erg Infect Dis 2003; 9:763-7. thermophilic amoebae and pathogenic Naegleria fowleri in a newly 27. Donlan RM. Biofilms: inicrobial life on surfaces. Emerg Infect Dis created cooling lake. Appl Environ Microbiol 1989; 55:722-32. 2002; 8:881-90. 12. Blair B, Sarkar P, Bright KR, Ivlarciano"Cabral F, Gerba CP. Naegleria 28. Marciano-Cabral F, Jamerson M, Kaneshiro ES. Free-living amoebae, fowleri in well water. En1erg Infect Dis 2008; 14:1499-501. Legionel/a and Mycobacterium in tap water supplies by a municipal 13. Kadlec V, Skv

Deaths Caused by Naeg/eria fawleri Infections • CID • 7