A preliminary study on Naegleria species in water bodies of Kurunegala district , Sri Lanka

Introduction and Objective: Species belonging to the genus Naegleria are free-living ubiquitous protozoa. They have been isolated from most regions of the world. N. fowleri causes an acute, fulminant and rapidly fatal infection involving the central nervous system (CNS) in humans. It is known as primary amoebic meningoencephalitis (PAM). Infection is generally acquired while swimming, diving and total submersion for bathing in freshwater-lakes and ponds. Many inland fresh water bodies are present in Sri Lanka. These water bodies are frequently used by people for their daily needs. However, studies have not yet been conducted to determine the prevalence of Naegleria species occurring in local water bodies. The present study was therefore, carried out to isolate Naegleria species from selected water bodies located in four Divisional Secretariat (DS) divisions in the Kurunegala district, Sri Lanka. Methods: Two different sites (clear and turbid water) of each tank were selected for sampling. Two water samples (surface water and deep water) were collected from each site (4 samples from one tank). Altogether, eighty water samples were collected from 20 tanks. Culture, enflagellation test and staining were done to detect Naegleria species. ArcGIS 10.3 and MINITAB (14) software were used for the data analysis. Results: Flagella transformation was observed in 19 (47.5%) surface water samples and 11 (27.5%) deep water samples. Of 20 tanks, 10 were positive for Naegleria species. Conclusions: Findings of the present study suggest that more specific genotyping studies are needed to confirm the presence of pathogenic N. fowleri in the study area.


Introduction
Naegleria species are free-living amoebae which inhabit warm fresh water bodies (rivers, lakes and hot springs) and soil. 1 There are several species in the Genus Naegleria.However, Naegleria fowleri is the only species known to cause infection in humans.It causes fulminant and rapidly fatal primary amoebic meningoencephalitis (PAM). 2 While PAM is a rare disease, it could be acquired while swimming, diving and total submersion for bathing in freshwaterlakes and ponds.Other Naegleria species such as N. australiensis and N. italic are known to cause PAM in experimentally infected animals only. 3In addition to PAM, humidifier fever can be caused by Naegleria spp. 4 It is a nonlethal hypersensitivity reaction caused due to antigenic material of Naegleria spp.present in humidifier systems.
Three distinct stages (amoeba, flagellate and cyst) of this organism can be identified in the life cycle. 5Trophozoites (10μm-25 μm) 6 multiply by binary fission 7 and encyst in response to unfavourable conditions. 8The cyst is round in shape with a single wall and size is varied (8μm-12 μm). 7udies have shown 92.9%, 35.3% and 15.0% prevalence of Naegleria species in environmental water samples, natural hot springs and recreational water in China, Thailand and Iran respectively. 9,10,11ere are many fresh water bodies (locally known as "tanks") in the dry zone of Sri Lanka.These tanks are primarily built for agricultural purposes.However, these water bodies are frequently used by people for their daily needs (mainly for washing and bathing).Conditions of these water bodies are ideal for the growth of Naegleria species.Except for one report 12 , the occurrence of Naegleria spp. in local water bodies has not yet been investigated.This preliminary study was therefore carried out to isolate Naegleria species from 20 water bodies of four Divisional Secretariat divisions in the Kurunegala district, Sri Lanka using culture techniques and the enflagellation test.

Study area
The Kurunegala district is located in the North-Western province of Sri Lanka.The total population of the study area was approximately 142,078 in a land area of 719 square kilometres 13 with a mean elevation of approximately 76 m from sea level.The annual rainfall is around 2,316.1 mm with the highest rainfall occurring in October and November during the North-East monsoon.The mean annual temperature is approximately 27.4  C and the annual relative humidity varies from 71-87%. 14e Kurunegala District consists of 30 Divisional Secretariat (DS) divisions from which the Maho, Nikaweratiya, Kotawehera and Abanpola DS divisions were selected for the study, based on the density of water-bodies.There are approximately 654 water bodies in the study area.A standard random number table was used to select the tanks.Of the 654 water bodies, 50 were randomly selected from the four DS Divisions.
As some of the randomly selected tanks were located in rural localities with no proper road access, 20 of the 50 tanks were selected for the study based on the convenience of collection and transportation of samples.

Collection of water sample
There were several entryways to a tank in the study area.Two different entryways were selected for sampling.The bathing area frequently used by people was selected as the first sampling site.A turbid area (frequented by cattle and buffaloes) was selected as the second sampling site.Two water samples were collected from each site (4 samples from one tank).One sample was collected from the surface water approximately 0.5 m away from the edge of the dam.The second water sample was collected by opening the lid of the container at a depth of 1m.The purpose of sampling from surface and deep water was to determine the presence of Naegleria species in either deep or surface water in the study area.Samples were collected during day time (12.00 noon to 14.00 pm) in sterile universal glass containers (30 ml).Sample collection was carried out during a warm and dry month (September 2016) of the year with a minimum rainfall.Containers were capped and labelled immediately after collection.The samples were transported at room temperature to the Department of Parasitology, Faculty of Medicine, University of Peradeniya for further investigations.

Global positioning system (GPS) coordinates
Geographical coordinates (Latitude and Longitudes) of sampling sites were recorded using an android GPS receiver.

Culture
The water samples were mixed well, and 15 ml of water transferred into new conical tubes.The tubes were centrifuged at 2000 rpm for 2 minutes (International centrifuge; GEC A4378x1).The supernatant was discarded, and the sediment was used for inoculation.Cultures were carried out on non-nutrient agar plates with Escherichia coli (NCTC10418) as described by Ash and Orihel (1987). 15Plates were sealed with parafilm to prevent contamination.Culture plates were incubated at 37  C overnight.The plates were observed for five consecutive days using an inverted microscope (Leitz Diavert).Positive growth was identified by increased localised amoebic count in the culture plate.Localised areas were marked on the plate.

Examination for flagellates (enflagellation test)
Scrapings from culture plates with growth were inoculated into 1 ml of distilled water and incubated at 37  C for 30 minutes.Wet smears were prepared and observed for transformation of trophozoites into pear shaped bi-flagellates or multi-flagellates. 16Preservation of flagellates was done using polyvinyl alcohol (PVA) fixative for trichrome staining.Trichrome stained trophozoites and flagellate forms were examined with a light microscope separately.Dimensions of trophozoites and flagellate forms were measured using a calibrated micrometer at (x100) magnification.

GIS analysis
GIS analysis was done using ArcGIS 10.3 software which works with maps to compile geographic information.

Statistical analysis
The results were analysed using MINITAB (14) statistical software.Two proportions and correlation tests were performed.Positivity in surface water vs deep water and positivity in first site vs second site were considered as variables.

Results
Eighty water samples were collected from 20 tanks.From each tank, sampling was done from two sites (clear and turbid water).From each site, two samples were collected (surface and deep).Flagella transformation was observed in 19 surface water samples (47.5%) and 11 (27.5%)deep water samples.Of 20 tanks, 10 (50%) were positive for Naegleria spp.(Table 1, 2 and Figure 1).There was no significant positive correlation (p>0.05) between clear and turbid water.However, we found a significant positive correlation between surface water and deep water (p<0.05).
The shape and size of the flagellate, trophozoite, and cyst were evaluated on trichrome stained slides for confirmation.
The trophozoites were active and constantly changing their size and shape.The length varied from 18-22 μm.The cytoplasm was finely granular and contained a conspicuous clear nuclear halo and a dense central nucleolus.Few vacuoles were visible in the cytoplasm.
The trophozoites were motile and moved by extending blunt rounded lobopodia (Figure 2).Biflagellates were pear-shaped (Figure 3).They were 10-12 μm in length and 5-7 μm in width.Flagella was fixed into the conical edge of the organism and showed sluggish circular movements.Multi-flagellates were not observed in cultured samples.Cysts were rounded and spherical in shape with smooth outer coverings.The length of the cyst varied between 6-9 μm.In most cases, the cytoplasm was granular (Figure 3).

Discussion
Epidemiologically, Naegleria has a worldwide distribution.Primary amoebic meningoencephalitis was first described by Malcolm Fowler in Australia. 17Thereafter, cases of PAM were identified in many parts of the world.Over 440 cases of PAM were reported worldwide.Among them, nearly half of the cases were reported from the United States. 18In Asia, the highest number of cases has been reported in India 19,20 followed by Pakistan. 21A single case of PAM has been reported due to freshwater pearl diving in Vietnam. 22e main method of contracting PAM is by swimming in N. fowleri contaminated fresh water collections.However, recent reports suggest that the use of "neti pots" for nasal irrigation can also predispose to PAM. 23 In addition; N. fowleri has been isolated from the nares of apparently healthy children. 24,25,26ither PAM nor N. fowleri has been reported in Sri Lanka.However, the climatic conditions prevailing in Sri Lanka is favourable for growth of Naegleria species in local water bodies.Thus, isolation and identification of N. fowleri in local water bodies is a necessity.However, identification of N. fowleri based on morphological characteristics is not easy due to the existence of several genera of amoebae with similar morphological features in the same ecological habitat. 27,28Furthermore, pathogenic N. fowleri and non-pathogenic Naegleria lovaniensis are antigenically related species. 28flagellation test (Amoeba-to-Flagellate Transformation) was performed to detect Naegleria species in the present study.However, the enflagellation test is not considered the gold standard test for detecting Naegleria species, because few non-flagellating Naegleria strains have been isolated from France. 29In addition, trophozoites isolated in one location of Australia have failed consistently to transform into flagellates.However, non-flagellating Naegleria species have not been reported so far in Asia or the Indian subcontinent.
Current methods for detection and enumeration of Naegleria species are based on culture techniques 30 followed by various other methods (species-specific monoclonal antibodies 31 , PCR 32 , enzyme electrophoresis 27 , isoenzyme electrophoretic profiles 33,34 and DNA restriction fragment length polymorphisms (RFLPs) 32,35,36 to identify the isolate up to the species level.Randomly amplified polymorphic DNA typing has been used to differentiate Naegleria spp.and could be used to detect minor variations in N. fowleri strains. 37In addition, N. fowleri positive cultures could be tested for pathogenicity by mice inoculation.
Records on the isolation of Naegleria from water bodies in tropical countries are very limited.Prevalence of 29% and 10% were reported in aquatic habitats in a human environment 38 and in stagnant water in an industrial area in Thailand. 39A recent study carried out in natural hot springs in 13 provinces of Thailand showed 35% positivity for Naegleria 10 indicating a high health risk to those exposed to such waters.Findings of our study also suggest a high prevalence (50%) of these free-living amoebae in aquatic habitats in the study area.These water-bodies form a network of reservoirs where water cascades and spills over to the tank situated below and finally drains into a common canal or stream.Hence, Naegleria can easily migrate from this network to other tanks.This could be one reason for the high prevalence of the organism in these lakes.
Usually in the dry season water is clear as no muddy rain water is drained to the tank.The most plausible reason for the turbid water in these tanks was the wallowing of agricultural animals (such as buffalos) for long hours in the water.These animals enter the tanks through specific entry points.These entry points are also used by the local people for bathing and various other purposes.The role of animals in Naegleria ecology and epidemiology is yet unclear.A study in Tennessee, USA, showed that 13 wild mammalian species had serum antibodies against Naegleria indicating past exposure 40 .This was the reason we compared sites in the tanks having both clear and turbid water.However, we could not find a significant difference (p>0.05) between the two sites, clear and turbid water.A study done in India showed 34.5% prevalence of Naegleria spp in surface water. 41It was 7.6% in deep well water in Arizona. 42e significant finding of this study was that the organism thrives more in surface water than in deep water (p<0.05).A similar result has been reported in some DS divisions in the same district previously. 43mitations: Small sample size and not performing specific tests (such as FAT or PCR) to detect Naegleria species were the limiting factors of this study.However, the findings of the present study would encourage more epidemiological investigations on Naegleria species occurring in local water bodies.

Conclusions
Our results show a high prevalence of Naegleria species in the water bodies of the study area.Consequently, there is a possibility of the existence of pathogenic Naegleria fowleri in these water bodies.More specific and accurate genotyping is required to confirm the presence of pathogenic Naegleria in these water bodies, as it may pose a health risk to people who use such water bodies for domestic and recreational activities.The findings of this study will be used in designing a long-term study for genotyping of isolates from different sites.

Figure 1 :
Figure 1: GIS map indicating sample sites and results of enflagellation test.

Table 1 :
Results of enflagellation test

Table 2 :
Sample positivity in DS Divisions