Evaluation of coccidia DNA in irrigation pond water and wastewater sludge associated with Cyclospora cayetanensis 18S rRNA gene qPCR detections.

The coccidian parasite Cyclospora cayetanensis is the causative agent for foodborne outbreaks of cyclosporiasis disease and multiple annual fresh produce recalls. The aim of this study was to identify potential cross-reacting species for the C. cayetanensis 18S rRNA and MIT1C gene target real-time quantitative polymerase chain reaction (qPCR) assays. The environmental samples evaluated were irrigation pond water, produce wash water, and wastewater treatment sludge from a previous study with qPCR detections of C. cayetanensis by the 18S rRNA gene target qPCR. From these samples, longer regions of the 18S rRNA gene and the mitochondrial cytochrome c oxidase subunit III gene (cox3) were sequenced. Of 65 irrigation pond water samples with positive test results using the C. cayetanensis 18S rRNA gene qPCR assay, none had MIT1C qPCR assay detections or sequences that clustered with C. cayetanensis based on sequencing of the cox3 and 18S rRNA gene. Sequences from these samples clustered around coccidia sequences found in bird, fish, reptile, and amphibian hosts. Of 26 sludge samples showing detections by either qPCR assay, 14 (54%) could be confirmed as containing C. cayetanensis by sequencing of cox3 and 18S rRNA gene regions. In three of the remaining sludge samples, sequenced reads clustered with coccidia from rodents. This study demonstrated that caution should be taken when interpreting qPCR C. cayetanensis detection data in environmental samples and sequencing steps will likely be needed for confirmation. IMPORTANCE: Fresh produce is a leading transmission source in cyclosporiasis outbreaks. It is therefore essential to understand the role that produce-growing environments play in the spread of this disease. To accomplish this, sensitive and specific tests for environmental and irrigation waters must be developed. Potential cross-reactions of Cyclospora cayetanensis real-time quantitative polymerase chain reaction (qPCR) assays have been identified, hindering the ability to accurately identify this parasite in the environment. Amplicon sequencing of the cox3 and 18S rRNA genes revealed that all irrigation pond water and two sludge samples that initially detected C. cayetanensis by qPCR were most likely cross-reactions with related coccidian organisms shed from birds, fish, reptiles, amphibians, and rodents. These results support that a single testing method for environmental samples is likely not adequate for sensitive and specific detection of C. cayetanensis.

Shiga Toxin-Producing Escherichia coli O157:H7 Illness Outbreak Associated with Untreated, Pressurized, Municipal Irrigation Water - Utah, 2023.

During July-September 2023, an outbreak of Shiga toxin-producing Escherichia coli O157:H7 illness among children in city A, Utah, caused 13 confirmed illnesses; seven patients were hospitalized, including two with hemolytic uremic syndrome. Local, state, and federal public health partners investigating the outbreak linked the illnesses to untreated, pressurized, municipal irrigation water (UPMIW) exposure in city A; 12 of 13 ill children reported playing in or drinking UPMIW. Clinical isolates were genetically highly related to one another and to environmental isolates from multiple locations within city A's UPMIW system. Microbial source tracking, a method to indicate possible contamination sources, identified birds and ruminants as potential sources of fecal contamination of UPMIW. Public health and city A officials issued multiple press releases regarding the outbreak reminding residents that UPMIW is not intended for drinking or recreation. Public education and UPMIW management and operations interventions, including assessing and mitigating potential contamination sources, covering UPMIW sources and reservoirs, indicating UPMIW lines and spigots with a designated color, and providing conspicuous signage to communicate risk and intended use might help prevent future UPMIW-associated illnesses.

Sources and Prevalence of Cyclospora cayetanensis in Southeastern U.S. Growing Environments.

Recent cyclosporiasis outbreaks associated with fresh produce grown in the United States highlight the need to better understand Cyclospora cayetanensis prevalence in U.S. agricultural environments. In this study, C. cayetanensis occurrence was assessed in municipal wastewater sludge, on-farm portable toilets, irrigation pond water, and spent packing house dump tank water in a Southeastern Georgia growing region over two years. Detection of the C. cayetanensis 18S rRNA qPCR gene target in pond samples was 0%, 28%, and 42% (N = 217) depending on the detection definition used, and ≤1% in dump tank samples (N = 46). However, no qPCR detections were confirmed by sequencing, suggesting false detection occurred due to cross-reactions. C. cayetanensis qPCR detections were confirmed in 9% of wastewater sludge samples (N = 76). The human-specific fecal markers HF183 and crAssphage were detected in 33% and 6% of pond samples, respectively, and 4% and 0% of dump tank samples, respectively. Despite community Cyclospora shedding and evidence of human fecal contamination in irrigation water, there was no correlation between C. cayetanensis and HF183 qPCR detections, further supporting that 18S gene target qPCR amplifications were due to cross-reactions. When evaluating C. cayetanensis qPCR environmental detection data, the impact of assay specificity and detection criteria should be considered. Moreover, additional sequence-based testing may be needed to appropriately interpret Cyclospora qPCR environmental data.

Naegleria fowleri Detected in Grand Teton National Park Hot Springs.

The free-living thermophilic amoeba Naegleria fowleri (N. fowleri) causes the highly fatal disease primary amoebic meningoencephalitis. The environmental conditions that are favorable to the growth and proliferation of N. fowleri are not well-defined, especially in northern regions of the United States. In this study, we used culture-based methods and multiple molecular approaches to detect and analyzeN. fowleri and other Naegleria spp. in water, sediment, and biofilm samples from five hot spring sites in Grand Teton National Park, Wyoming, U.S.A. These results provide the first detections of N. fowleri in Grand Teton National Park and provide new insights into the distribution of pathogenic N. fowleri and other nonpathogenic Naegleria spp. in natural thermal water systems in northern latitudes.

A Case of Primary Amebic Meningoencephalitis Associated with Surfing at an Artificial Surf Venue: Environmental Investigation.

Naegleria fowleri is a thermophilic ameba found in freshwater that causes primary amebic meningoencephalitis (PAM) when it enters the nose and migrates to the brain. In September 2018, a 29-year-old man died of PAM after traveling to Texas. We conducted an epidemiologic and environmental investigation to identify the water exposure associated with this PAM case. The patient's most probable water exposure occurred while surfing in an artificial surf venue. The surf venue water was not filtered or recirculated; water disinfection and water quality testing were not documented. N. fowleri and thermophilic amebae were detected in recreational water and sediment samples throughout the facility. Codes and standards for treated recreational water venues open to the public could be developed to address these novel venues. Clinicians and public health officials should also consider novel recreational water venues as a potential exposure for this rare amebic infection.

Microbial Characterization, Factors Contributing to Contamination, and Household Use of Cistern Water, U.S. Virgin Islands.

Households in the United States Virgin Islands (USVI) heavily rely on roof-harvested rainwater stored in cisterns for their daily activities. However, there are insufficient data on cistern water microbiological and physicochemical characteristics to inform appropriate cistern water management. Cistern and kitchen tap water samples were collected from 399 geographically representative households across St. Croix, St. Thomas, and St. John and an administered survey captured household site and cistern characteristics and water use behaviors. Water samples were analyzed for Escherichia coli by culture, and a subset of cistern water samples (N = 47) were analyzed for Salmonella, Naegleria fowleri, pathogenic Leptospira, Cryptosporidium, Giardia, and human-specific fecal contamination using real-time polymerase chain reaction (PCR). Associations between E. coli cistern contamination and cistern and site characteristics were evaluated to better understand possible mechanisms of contamination. E. coli was detected in 80% of cistern water samples and in 58% of kitchen tap samples. For the subset of samples tested by PCR, at least one of the pathogens was detected in 66% of cisterns. Our results suggest that covering overflow pipes with screens, decreasing animal presence at the household, and preventing animals or insects from entering the cisterns can decrease the likelihood of E. coli contamination in USVI cistern water.

Outbreaks of Acute Gastrointestinal Illness Associated with a Splash Pad in a Wildlife Park - Kansas, June 2021.

In June 2021, Kansas state and county public health officials identified and investigated three cases of shigellosis (a bacterial diarrheal illness caused by Shigella spp.) associated with visiting a wildlife park. The park has animal exhibits and a splash pad. Two affected persons visited animal exhibits, and all three entered the splash pad. Nonhuman primates are the only known animal reservoir of Shigella. The splash pad, which sprays water on users and is designed so that water does not collect in the user area, was closed on June 19. The state and county public health codes do not include regulations for splash pads. Thus, these venues are not typically inspected, and environmental health expertise is limited. A case-control study identified two distinct outbreaks associated with the park (a shigellosis outbreak involving 21 cases and a subsequent norovirus infection outbreak involving six cases). Shigella and norovirus can be transmitted by contaminated water; in both outbreaks, illness was associated with getting splash pad water in the mouth (multiply imputed adjusted odds ratio [aORMI] = 6.4, p = 0.036; and 28.6, p = 0.006, respectively). Maintaining adequate water disinfection and environmental health expertise and targeting prevention efforts to caregivers of splash pad users help prevent splash pad-associated outbreaks. Outbreak incidence might be further reduced when U.S. jurisdicitons voluntarily adopt CDC's Model Aquatic Health Code (MAHC) recommendations and through the prevention messages: "Don't get in the water if sick with diarrhea," "Don't stand or sit above the jets," and "Don't swallow the water."†.

A Cross-Sectional Household Survey in the US Virgin Islands (2019) Reveals Cisterns as Challenging Peridomestic Aedes aegypti Habitats.

Most residences in the United States Virgin Islands (USVI) rely on household rainwater-catchment systems and subterranean cisterns for long-term water storage that may provide suitable habitats for mosquitoes of public health relevance. We conducted a household cistern survey (n = 164) on the islands of St. Croix, St. John, and St. Thomas in 2019. The survey revealed that 45.7% (95% CI: 38.3-53.4%) of cisterns contained mosquitoes (adult and/or immature mosquitoes). Aedes aegypti, a vector of chikungunya, dengue, and Zika viruses in the USVI, was found in 27.4% (95% CI: 21.2-34.7%) of cisterns and accounted for 83.3% of the total mosquitoes identified in the study. The odds of detecting mosquitoes in a cistern were 5.45 times higher at locations where the residents reported that they had observed adult mosquitoes coming out of their cisterns (95% CI: 2.25-14.21), suggesting that vector control personnel should consider resident complaints about mosquitoes in their cistern as valid and likely reliable self-assessments. Resident mosquito management practices in cisterns did not correspond with decreased odds of mosquito detection. We conclude that cisterns in the USVI commonly provide habitat for immature and adult Ae. aegypti, which may decrease the effectiveness of area-wide mosquito control strategies. Additional studies are necessary to evaluate the importance of these cisterns as they relate to mosquito production and arbovirus transmission risk, and to assess physical and chemical control methods.

Detection of Cyclospora cayetanensis in produce irrigation and wash water using large-volume sampling techniques.

The recent increase of reported cyclosporiasis outbreaks associated with fresh produce has highlighted the need for understanding environmental transmission of Cyclospora cayetanensis in agricultural settings and facilities. Conducting such environmental investigations necessitates robust sample collection and analytical methods to detect C. cayetanensis in water samples. This study evaluated three sample collection methods for recovery of C. cayetanensis oocysts from water samples during seeded recovery experiments. Two filtration-based methods, dead-end ultrafiltration (DEUF) and USEPA Method 1623.1, were evaluated for oocyst recovery from irrigation water. A non-filter-based method, continuous flow centrifugation (CFC), was evaluated separately for recovery from creek water and spent produce wash water. Median C. cayetanensis recovery efficiencies were 17% for DEUF and 16-22% for Method 1623.1. The DEUF method proved to be more robust than Method 1623.1, as the recovery efficiencies were less variable and the DEUF ultrafilters were capable of filtering larger volumes of high-turbidity water without clogging. Median C. cayetanensis recovery efficiencies for CFC were 28% for wash water and 63% for creek water, making it a viable option for processing water with high turbidity or organic matter. The data from this study demonstrate the capability of DEUF and CFC as filter-based and non-filter-based options, respectively, for the recovery of C. cayetanensis oocysts from environmental and agricultural waters.

Identifying septic pollution exposure routes during a waterborne norovirus outbreak - A new application for human-associated microbial source tracking qPCR.

In June 2017, the Pennsylvania Department of Health (PADOH) was notified of multiple norovirus outbreaks associated with 179 ill individuals who attended separate events held at an outdoor venue and campground over a month period. Epidemiologic investigations were unable to identify a single exposure route and therefore unable to determine whether there was a persistent contamination source to target for exposure mitigation. Norovirus was detected in a fresh recreational water designated swimming area and a drinking water well. A hydrogeological site evaluation suggested a nearby septic leach field as a potential contamination source via ground water infiltration. Geological characterization revealed a steep dip of the bedrock beneath the septic leach field toward the well, providing a viral transport pathway in a geologic medium not previously documented as high risk for viral ground water contamination. The human-associated microbial source tracking (MST) genetic marker, HF183, was used as a microbial tracer to demonstrate the hydrogeological connection between the malfunctioning septic system, drinking water well, and recreational water area. Based on environmental investigation findings, venue management and local public health officials implemented a series of outbreak prevention strategies including discontinuing the use of the contaminated well, issuing a permit for a new drinking water well, increasing portable toilet and handwashing station availability, and promoting proper hand hygiene. Despite the outbreaks at the venue and evidence of ground water contamination impacting nearby recreational water and the drinking water well, no new norovirus cases were reported during a large event one week after implementing prevention practices. This investigation highlights a new application for human-associated MST methods to trace hydrological connections between multiple fecal pollutant exposure routes in an outbreak scenario. In turn, pollutant source information can be used to develop effective intervention practices to mitigate exposure and prevent future outbreaks associated with human fecal contaminated waters.

Detection of Cryptosporidium Recovered from Large-Volume Water Samples Using Dead-End Ultrafiltration.

The procedure described here provides instructions for detection of Cryptosporidium recovered from large-volume water samples. Water samples are collected by dead-end ultrafiltration in the field and ultrafilters are processed in a laboratory. Microbes recovered from the filters are further concentrated and subjected to Cryptosporidium isolation or nucleic acid extraction methods for the detection of Cryptosporidium oocysts or Cryptosporidium DNA.

Shiga Toxin-Producing Escherichia coli Infections Associated With Romaine Lettuce-United States, 2018.

BACKGROUND: Produce-associated outbreaks of Shiga toxin-producing Escherichia coli (STEC) were first identified in 1991. In April 2018, New Jersey and Pennsylvania officials reported a cluster of STEC O157 infections associated with multiple locations of a restaurant chain. The Centers for Disease Control and Prevention (CDC) queried PulseNet, the national laboratory network for foodborne disease surveillance, for additional cases and began a national investigation. METHODS: A case was defined as an infection between 13 March and 22 August 2018 with 1 of the 22 identified outbreak-associated E. coli O157:H7 or E. coli O61 pulsed-field gel electrophoresis pattern combinations, or with a strain STEC O157 that was closely related to the main outbreak strain by whole-genome sequencing. We conducted epidemiologic and traceback investigations to identify illness subclusters and common sources. A US Food and Drug Administration-led environmental assessment, which tested water, soil, manure, compost, and scat samples, was conducted to evaluate potential sources of STEC contamination. RESULTS: We identified 240 case-patients from 37 states; 104 were hospitalized, 28 developed hemolytic uremic syndrome, and 5 died. Of 179 people who were interviewed, 152 (85%) reported consuming romaine lettuce in the week before illness onset. Twenty subclusters were identified. Product traceback from subcluster restaurants identified numerous romaine lettuce distributors and growers; all lettuce originated from the Yuma growing region. Water samples collected from an irrigation canal in the region yielded the outbreak strain of STEC O157. CONCLUSIONS: We report on the largest multistate leafy greens-linked STEC O157 outbreak in several decades. The investigation highlights the complexities associated with investigating outbreaks involving widespread environmental contamination.

Response and remediation actions following the detection of Naegleria fowleri in two treated drinking water distribution systems, Louisiana, 2013-2014.

Naegleria fowleri causes the usually fatal disease primary amebic meningoencephalitis (PAM), typically in people who have been swimming in warm, untreated freshwater. Recently, some cases in the United States were associated with exposure to treated drinking water. In 2013, a case of PAM was reported for the first time in association with the exposure to water from a US treated drinking water system colonized with culturable N. fowleri. This system and another were found to have multiple areas with undetectable disinfectant residual levels. In response, the water distribution systems were temporarily converted from chloramine disinfection to chlorine to inactivate N. fowleri and reduced biofilm in the distribution systems. Once >1.0 mg/L free chlorine residual was attained in all systems for 60 days, water testing was performed; N. fowleri was not detected in water samples after the chlorine conversion. This investigation highlights the importance of maintaining adequate residual disinfectant levels in drinking water distribution systems. Water distribution system managers should be knowledgeable about the ecology of their systems, understand potential water quality changes when water temperatures increase, and work to eliminate areas in which biofilm growth may be problematic and affect water quality.

First Reported Human Cases of Leptospirosis in the United States Virgin Islands in the Aftermath of Hurricanes Irma and Maria, September-November 2017.

OBJECTIVE: Following Hurricanes Irma and Maria, the first case of human leptospirosis ever identified in the US Virgin Islands (USVI) was reported to the Virgin Islands Department of Health. Leptospirosis is a potentially fatal bacterial disease caused by Leptospira species found in animal urine and urine-contaminated water and soil. Outbreaks can occur following extreme weather events. METHOD: Additional cases of leptospirosis were identified in the 2.5 months post-hurricanes by reviewing emergency department (ED) records from territorial hospitals for patients demonstrating leptospirosis-consistent symptoms, testing symptomatic patients previously enrolled in the USVI arbovirus surveillance system (VIASS), and adding leptospirosis testing prospectively to VIASS. Available patient sera underwent local rapid diagnostic testing for anti-Leptospira IgM followed by confirmatory microscopic agglutination testing at the US Centers for Disease Control and Prevention. Water was collected from cisterns with epidemiologic links to confirmed cases and tested by real-time PCR (qPCR) for pathogenic Leptospira spp. RESULTS: Sixteen retrospectively identified symptomatic patients were enrolled in VIASS; 15 with available samples tested negative. Based on review of 5226 ED charts, 6 patients were further investigated; of these, 5 were tested of which 1 was positive. Prospective leptospirosis surveillance tested 57 additional patients; of these, 1 was positive. Water from 1 of 5 tested cisterns was found positive by qPCR. CONCLUSIONS: This investigation documents the first 3 cases of leptospirosis reported in the USVI and demonstrates how VIASS successfully was adapted to establish leptospirosis surveillance. Contaminated cistern water was identified as a potential source for Leptospira spp. transmission, highlighting the need for additional post-hurricane remediation and disinfection guidance.

Outbreaks Associated with Untreated Recreational Water - United States, 2000-2014.

Outbreaks associated with untreated recreational water can be caused by pathogens, toxins, or chemicals in fresh water (e.g., lakes, rivers) or marine water (e.g., ocean). During 2000-2014, public health officials from 35 states and Guam voluntarily reported 140 untreated recreational water-associated outbreaks to CDC. These outbreaks resulted in at least 4,958 cases of disease and two deaths. Among the 95 outbreaks with a confirmed infectious etiology, enteric pathogens caused 80 (84%); 21 (22%) were caused by norovirus, 19 (20%) by Escherichia coli, 14 (15%) by Shigella, and 12 (13%) by Cryptosporidium. Investigations of these 95 outbreaks identified 3,125 cases; 2,704 (87%) were caused by enteric pathogens, including 1,459 (47%) by norovirus, 362 (12%) by Shigella, 314 (10%) by Cryptosporidium, and 155 (5%) by E. coli. Avian schistosomes were identified as the cause in 345 (11%) of the 3,125 cases. The two deaths were in persons affected by a single outbreak (two cases) caused by Naegleria fowleri. Public parks (50 [36%]) and beaches (45 [32%]) were the leading settings associated with the 140 outbreaks. Overall, the majority of outbreaks started during June-August (113 [81%]); 65 (58%) started in July. Swimmers and parents of young swimmers can take steps to minimize the risk for exposure to pathogens, toxins, and chemicals in untreated recreational water by heeding posted advisories closing the beach to swimming; not swimming in discolored, smelly, foamy, or scummy water; not swimming while sick with diarrhea; and limiting water entering the nose when swimming in warm freshwater.

Outbreaks Associated with Treated Recreational Water - United States, 2000-2014.

Outbreaks associated with exposure to treated recreational water can be caused by pathogens or chemicals in venues such as pools, hot tubs/spas, and interactive water play venues (i.e., water playgrounds). During 2000-2014, public health officials from 46 states and Puerto Rico reported 493 outbreaks associated with treated recreational water. These outbreaks resulted in at least 27,219 cases and eight deaths. Among the 363 outbreaks with a confirmed infectious etiology, 212 (58%) were caused by Cryptosporidium (which causes predominantly gastrointestinal illness), 57 (16%) by Legionella (which causes Legionnaires' disease, a severe pneumonia, and Pontiac fever, a milder illness with flu-like symptoms), and 47 (13%) by Pseudomonas (which causes folliculitis ["hot tub rash"] and otitis externa ["swimmers' ear"]). Investigations of the 363 outbreaks identified 24,453 cases; 21,766 (89%) were caused by Cryptosporidium, 920 (4%) by Pseudomonas, and 624 (3%) by Legionella. At least six of the eight reported deaths occurred in persons affected by outbreaks caused by Legionella. Hotels were the leading setting, associated with 157 (32%) of the 493 outbreaks. Overall, the outbreaks had a bimodal temporal distribution: 275 (56%) outbreaks started during June-August and 46 (9%) in March. Assessment of trends in the annual counts of outbreaks caused by Cryptosporidium, Legionella, or Pseudomonas indicate mixed progress in preventing transmission. Pathogens able to evade chlorine inactivation have become leading outbreak etiologies. The consequent outbreak and case counts and mortality underscore the utility of CDC's Model Aquatic Health Code (https://www.cdc.gov/mahc) to prevent outbreaks associated with treated recreational water.

Cholera Epidemic - Lusaka, Zambia, October 2017-May 2018.

On October 6, 2017, an outbreak of cholera was declared in Zambia after laboratory confirmation of Vibrio cholerae O1, biotype El Tor, serotype Ogawa, from stool specimens from two patients with acute watery diarrhea. The two patients had gone to a clinic in Lusaka, the capital city, on October 4. Cholera cases increased rapidly, from several hundred cases in early December 2017 to approximately 2,000 by early January 2018 (Figure). In collaboration with partners, the Zambia Ministry of Health (MoH) launched a multifaceted public health response that included increased chlorination of the Lusaka municipal water supply, provision of emergency water supplies, water quality monitoring and testing, enhanced surveillance, epidemiologic investigations, a cholera vaccination campaign, aggressive case management and health care worker training, and laboratory testing of clinical samples. In late December 2017, a number of water-related preventive actions were initiated, including increasing chlorine levels throughout the city's water distribution system and placing emergency tanks of chlorinated water in the most affected neighborhoods; cholera cases declined sharply in January 2018. During January 10-February 14, 2018, approximately 2 million doses of oral cholera vaccine were administered to Lusaka residents aged ≥1 year. However, in mid-March, heavy flooding and widespread water shortages occurred, leading to a resurgence of cholera. As of May 12, 2018, the outbreak had affected seven of the 10 provinces in Zambia, with 5,905 suspected cases and a case fatality rate (CFR) of 1.9%. Among the suspected cases, 5,414 (91.7%), including 98 deaths (CFR = 1.8%), occurred in Lusaka residents.

Primary Amebic Meningoencephalitis Associated With Rafting on an Artificial Whitewater River: Case Report and Environmental Investigation.

Background: Naegleria fowleri is a thermophilic ameba found in freshwater that causes primary amebic meningoencephalitis (PAM) when it enters the nose and migrates to the brain. Patient exposure to water containing the ameba typically occurs in warm freshwater lakes and ponds during recreational water activities. In June 2016, an 18-year-old woman died of PAM after traveling to North Carolina, where she participated in rafting on an artificial whitewater river. Methods: We conducted an epidemiologic and environmental investigation to determine the water exposure that led to the death of this patient. Results: The case patient's most probable water exposure occurred while rafting on an artificial whitewater river during which she was thrown out of the raft and submerged underwater. The approximately 11.5 million gallons of water in the whitewater facility were partially filtered, subjected to ultraviolet light treatment, and occasionally chlorinated. Heavy algal growth was noted. Eleven water-related samples were collected from the facility; all were positive for N. fowleri. Of 5 samples collected from the nearby natural river, 1 sediment sample was positive for N. fowleri. Conclusions: This investigation documents a novel exposure to an artificial whitewater river as the likely exposure causing PAM in this case. Conditions in the whitewater facility (warm, turbid water with little chlorine and heavy algal growth) rendered the water treatment ineffective and provided an ideal environment for N. fowleri to thrive. The combination of natural and engineered elements at the whitewater facility created a challenging environment to control the growth of N. fowleri.