Surveillance of Naegleria fowleri in Louisiana's public water systems

Naegleria fowleri, commonly called the ‘brain-eating amoeba’, is a pathogenic free-living amoeba (FLA), which is found naturally in hot springs and warm surface waters. N. fowleri causes primary amoebic meningoencephalitis (PAM). This infection occurs when the N. fowleri enters the nasal cavity, travels through the olfactory mucosa and along the olfactory nerve, and ultimately feeds on nerve tissues in the brain, resulting in tissue necrosis, bleeding, and – in nearly all cases – death within 2 weeks of initial infection. Three deaths that were attributed to PAM have been recorded in recent years in Louisiana (Isaac & Sherchan 2020).

N. fowleri is listed in the United States Environmental Protection Agency (U.S. EPA) draft Contaminant Candidate List 5 (CCL5). However, less frequently known is that N. fowleri amoeba can occur in treated waters such as swimming pools, water heaters, municipal systems, and other water transport systems that have been inadequately treated or negligently maintained to prevent amoebic growth. It is possible to contract the amoeba through treated water with no or low chlorine residual. There have been several recent cases of N. fowleri infections via municipal water sources (Yu et al. 2017). In 2011, two people died of PAM caused by N. fowleri in DeSoto and St. Bernard Parishes in Louisiana. Both cases involved treated tap water that lacked detectable chlorine residual that was self-introduced into the sinus cavity via net-pots. In 2013, there was a second death in St. Bernard Parish (a 4-year-old boy) caused by PAM, which was confirmed to be N. fowleri infection. The route of exposure was inhalation of treated tap water while the child played on a home Slip n’ Slide (water slide). Four out of 16 water distribution system samples collected by the CDC in St. Bernard Parish were positive for N. fowleri. According to Cope et al. (2019), the water temperature was >30 °C in three of the four positive sampling locations. To date, a total of nine Louisiana public water systems have tested positive for N. fowleri.

The Louisiana Department of Health (LDH) also confirmed the presence of N. fowleri in South Bossier Parish on 28 September 2018 (Wooten 2019). N. fowleri is thermophilic and able to proliferate in temperatures up to 45 °C. Warmer surface water temperatures due to global climate change will provide new environmental niches and higher risks of N. fowleri exposure (Bright & Gerba 2017; Xue et al. 2018). The annual number of N. fowleri cases associated with recreational and tap water exposure is increasing globally (Yu et al. 2017; Xue et al. 2018; Cope et al. 2019). However, water utilities only test for indicator organisms and are currently not required to test for the presence of N. fowleri due to limitations related to federal regulations, cost, time, and labor. In this study, we summarized N. fowleri surveillance in potable water systems in the State of Louisiana.

Ultrafilters that had processed 100 L of water samples were immediately transported to the laboratory in a cooler. These ultrafilters were backlashed as described by Cope et al. (2019) using WB saline with 0.1% Tween 80 and eluates processed by centrifugation at 1,500 g for 15 min at room temperature. Supernatants were removed and then pellets that were mixed with an overnight culture of Escherichia coli were plated on Nelson's agar and incubated at 42 °C for up to 7 days. Then, plates were examined using an inverted microscope to observe amoeba activity. At 7 days of incubation, the entire surface of the plate was harvested and centrifuged to produce a pellet, and DNA was extracted for quantitative polymerase chain reaction (qPCR) confirmation (Cope et al. 2019).

The TaqMan-based assay was performed with 5 μL of template DNA, 250 nM of the forward (JBVF, 5′-AGG TAC TTA CGT TAG AGT GCT AGT-3′) and reverse primers (JBVR, 5′-ATG GGA CAA TCC GGT TTT CTC A-3′), 100 nM of the FAM-labeled probe (JBVP, 5′-FAM-AC GCC CTA GCT GGT TAT GCC GGA TT-BHQ1-3′), and nanopure water (Mull et al. 2013). The thermal cycling conditions of the qPCR assay were as follows: (i) 95 °C for 15 min (activation of Taq DNA polymerase) and (ii) 45 cycles of 95 °C for 15 s and 63 °C for 33 s (Mull et al. 2013). No genotyping was performed in this study and organisms obtained by culture were confirmed only by qPCR for N. fowleri as described by Cope et al. (2015).

A total of 93 samples were collected during the period 2014–2018 and consisted of raw source water; point of entry (POE) – treated water delivered to the distribution; total coliform rule (TCR) – distribution system lines; additional chlorine residual (ACR) – distribution system lines; maximum residence time (MRT) – oldest water in the distribution system.

However, the results presented need to be interpreted with care. First, not all water systems have been sampled in each of those parishes. Second, most water systems only serve water to certain sections of the parish (not the whole parish). For example, St. John WD1, Ebarb WD1, Ascension CUD1, Schriever, North Monroe, and the City of Bossier only serve water to a portion of the parish, unlike St. Bernard that serves the whole parish. Of the parishes with N. fowleri-positive water samples, Ouachita Parish had the greatest number, with eight positive samples (two raw water samples and six distribution system samples). Five positive samples were from St. Bernard Parish, including four distribution system samples and one raw water sample. In Terrebonne Parish, three positive samples were two from distribution systems and one raw water sample. Two positive samples were also in Sabine Parish – one of these was raw water samples, while the other was from a distribution system. Ascension Parish had two positive samples, including one raw water sample and one distribution system sample, while Bossier Parish also had two positive samples, both from distribution systems. The remaining five parishes each had one positive sample – from a distribution system in St. John Parish and from raw water in St. Mary, Tensas, Caldwell, and St. James parishes. Of all positive samples, 10 raw source water samples tested positive for the presence of N. fowleri and 17 distribution system samples tested positive (Table 1).

Deaths from PAM contracted through drinking water distribution systems have occurred in many countries, including Australia, the United States, and Pakistan (Miller et al. 2018). Generally, drinking water sources are treated with chlorine, and in the case of Australia, the target concentration was 0.5 mg/L, which was constantly maintained throughout the entire system and appears to control the growth of N. fowleri (Morgan et al. 2016). However, a study found that N. fowleri (presumably as cysts) can survive short durations of higher concentrations of chlorine, even up to 20 mg/L (Miller et al. 2015). In Louisiana, following the St. Bernard 2013 PAM infection, LDH issued a new rule requiring public water systems to maintain a minimum of 0.5 mg/L of free or total chlorine throughout the entire drinking water system constantly and must be monitored regularly. However, seven out of 27 water systems tested in Louisiana were found to contain N. fowleri mostly at locations with low (0.5 mg/L) to no chlorine (LDHH 2017).

N. fowleri has a three-stage lifecycle. N. fowleri exists in soil in a cystic form and excysts to a flagellate stage when in contact with warm water and a source of food (bacteria). The trophozoite stage feeds on bacteria at the air– or biofilm–water interface in hot springs, surface waters, and other water systems that may remain still for prolonged periods (Mull et al. 2013; Cope & Ali 2016; Lu et al. 2016). To date, a total of 145 positive cases of PAM caused by N. fowleri have been reported in the United States (CDC 2019; Gharpure et al. 2021). The majority of N. fowleri cases (over >50%) in the United States have occurred in Texas and Florida from 1962 to 2008 (Yoder et al. 2010). However, recent cases have also occurred in north-tier states like Minnesota (2012), Virginia, Kansas, and North Carolina (2016) (Gharpure et al. 2021). Moreover, as changes in the global climate result in warmer surface temperatures, infections by N. fowleri are expected to increase (Gharpure et al. 2021). Further studies would generate a robust dataset on the impact of changing climate on the proliferation of N. fowleri in natural and engineered water systems.

Our research demonstrates that N. fowleri was detected in 27 samples out of 93 (29%) during the period 2014–2018. More studies are needed to find a better indicator for N. fowleri. Since 10 raw source water and 17 distribution system water samples tested positive, it is important to manage PAM risk by monitoring residual chlorine at the end of distribution system lines, as required by the LDH.

We thank Caryn Benjamin and Amanda Ames from the Department of Health and Hospitals, Baton Rouge, Louisiana for their contributions.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.