The effectiveness of water treatment processes against schistosome cercariae: A systematic review

Abstract

Background

Schistosomiasis is one of the most disabling neglected tropical diseases, ranking second in terms of years lived with disability. While treatment with the drug praziquantel can have immediate beneficial effects, reinfection can occur rapidly if people are in contact with cercaria-infested water. Water treatment for schistosomiasis control seeks to eliminate viable cercariae from water, thereby providing safe alternative water supplies for recreational and domestic activities including laundry and bathing. This provision may reduce contact with infested water, which is crucial for reducing reinfection following chemotherapy and cutting schistosome transmission.

Methodology

A qualitative systematic review was carried out to summarize the existing knowledge on the effectiveness of water treatment in removing or inactivating human schistosome cercariae. Four online databases were searched. Studies were screened and categorized into five water treatment processes: storage, heating, chlorination, filtration, and ultraviolet (UV) disinfection.

Conclusions

All five water treatment methods can remove or inactivate cercariae in water, and hence produce cercaria-free water. However, reliable design guidelines for treating water do not exist as there are insufficient data. Overall, the review found that cercariae are inactivated when storing water for 10–72 hours (depending on temperature), or with chlorination values of 3–30 mg-min/l. UV fluences between 3–60 mJ/cm2 may significantly damage or kill cercariae, and sand filters with 0.18–0.35 mm grain size have been shown to remove cercariae. This systematic review identified 67 studies about water treatment and schistosomiasis published in the past 106 years. It highlights the many factors that influence the results of water treatment experiments, which include different water quality conditions and methods for measuring key parameters. Variation in these factors limit comparability, and therefore currently available information is insufficient for providing complete water treatment design recommendations.

About the Authors:

Laura Braun

Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – original draft

* E-mail: [email protected]

Affiliation: Department of Civil and Environmental Engineering, South Kensington Campus, Imperial College London, London, United Kingdom

ORCID http://orcid.org/0000-0001-9319-0859

Jack E. T. Grimes

Roles Conceptualization, Data curation, Investigation, Methodology, Validation, Writing – review & editing

Affiliation: Department of Civil and Environmental Engineering, South Kensington Campus, Imperial College London, London, United Kingdom

ORCID http://orcid.org/0000-0002-9001-446X

Michael R. Templeton

Roles Conceptualization, Funding acquisition, Methodology, Supervision, Writing – review & editing

Affiliation: Department of Civil and Environmental Engineering, South Kensington Campus, Imperial College London, London, United Kingdom

Introduction

Schistosomiasis is a water-borne helminthic disease caused by schistosomes, which are parasitic worms. Infection occurs through dermal-contact with cercaria-infested freshwater. Cercariae are released by snails infected with miracidia which hatch from the eggs in human urine and feces. Despite efforts to control the disease, it remains a major public health problem. Disability-Adjusted Life Year (DALY) estimates have in fact increased over the past 20 years, exceeding 3.3 million DALYs according to the Global Burden of Disease Study 2010 [1, 2]. In 2012, the World Health Assembly (WHA) adopted resolution 65.21 aiming for the elimination of schistosomiasis. This resolution emphasizes the need for multi-sectoral rationale including chemotherapy, strengthening health systems, WASH, and snail control. The need for an integral approach to tackling schistosomiasis has been echoed by many researchers [3–8].

Schistosome cercariae are approximately 500 μm in length, up to 64 μm in width, and are released into freshwater by intermediate host snails [9, 10]. These snails can shed hundreds of cercariae a day–approximately 200 for S. haematobium, 250–600 for S. mansoni, and 15–160 for S. japonicum [11, 12]. Schistosome cercariae are attracted to the human host through skin chemicals, temperature gradient, and turbulence [13, 14]. They are non-feeders, and depend on endogenous glycogen reserves [15]. Cercariae have a forked tail which they use to propel forward and penetrate the host skin. Inside the host, cercariae develop into schistosomula, which eventually develop into schistosomes.

The full schistosome lifecycle was first described in 1908 [16], and interest grew subsequently as troops fighting on African territory were becoming infected [17]. Initially, the control measures focused on mollusciciding and preventing contact with contaminated water (such as described in the War Memoranda from 1919 [18]). The enthusiasm for snail control led to the development of molluscicides (pesticides against snails), which were used to kill all schistosome lifecycle stages in water [19]. The research on water treatment and snail control was slowed by the development of effective orally-administered drugs in the late 1970’s [17, 19]. Ever since, chemotherapy has been the focus of schistosomiasis control programs [8].

The schistosome lifecycle can be cut through chemotherapy, intermediate host control, and WASH. The orally administered drug praziquantel is used to treat schistosomiasis by killing adult worms in the human host. However, it does not prevent reinfection. Snail control reduces the number of intermediate host snails, and is commonly applied through mollusciciding. Although snail control has been shown to reduce disease burden, there are environmental risks associated with molluscicides [19–21]. Universal sanitation aims to prevent eggs found in human feces from entering water bodies, thereby lowering the number of miracidia. However, Grimes et al. found no significant association between school sanitation adequacy and S. mansoni infection intensity in a national survey in Ethiopia [22]. The positive effect of sanitation may only be realized when the entire community adopts the sanitation infrastructure; one miracidium is sufficient to infect a snail, which may release up to 20,000 cercariae in its lifetime, and as a result, only few miracidia are needed to maintain the schistosome lifecycle [12]. Increased hygiene may also impact schistosomiasis control due to the toxicity of soaps to miracidia and cercariae. Use of soaps has been linked with lower prevalence, especially in women who are more exposed to activities such as doing laundry which use soap [23]. Similarly, access to safe water has been found to reduce schistosomiasis prevalence (safe water implying cercaria-free water), as it reduces skin contact with contaminated water [24–26].

The options for providing cercaria-free water are either treating the water, or providing an alternate safe source of water (such as boreholes or rainwater). In endemic regions, there is often no safe alternative water source, and hence water treatment is required to provide a safe water supply. Access to safe water reduces the contact with cercaria-infested water, and may also reduce the risk of water being contaminated with human excreta, as people are spending less time at transmission sites.

Water treatment can provide safe water for domestic use such as washing, laundry, and bathing—activities which are associated with long water-contact periods, and therefore higher risk of schistosomiasis contraction. The community may still have to collect water from transmission sites in order to treat it, but water collection only exposes small areas of the body for short periods and is therefore likely to be less critical. Providing a community with water treatment infrastructure may consequently remove household and recreational activities from transmission sites. This has been demonstrated by numerous studies which observed significantly lower infection rates following the installation of water treatment plants or water recreation areas [24–27]. Water treatment infrastructure can also provide safe water for animals which act as reservoir hosts, particularly for S. japonicum, and which can play a significant part in transmission [28, 29].

Water treatment infrastructure should not replace other interventions, such as preventive chemotherapy or snail control. On the contrary, it should be implemented alongside chemotherapy-based and behavioral change programs to reduce water contact and thereby minimize the likelihood of reinfection following drug administration. Occupational activities such as fishing and sand harvesting rely on natural water bodies, and exposure to contaminated water will not be affected by the introduction of a safe water supply. Alternative preventive measures may be needed to reduce such forms of exposure (e.g. boots and gloves for fishermen). Even with the provision of safe water, the community may continue to access transmission sites for reasons including preference for untreated water, overcrowding, or lack of privacy at the safe water source, so health education and campaigns to raise awareness of the dangers of exposure to infested water should always accompany water infrastructure implementation. While eliminating contact with infested water bodies completely and forever may be unachievable, strategies could at least be targeted at reducing this contact during and after PC programs, to reduce reinfection rates and hopefully allow a move towards complete elimination of infection in the community.

Water treatment infrastructure also needs careful planning to ensure the technology is sustainable and acceptable to the community. Firstly, however, research is needed to determine which water treatment processes are effective at removing or inactivating cercariae, which is the motivation for this systematic review.

Methods

The systematic review follows the guidelines of the Preferred Reporting Items for Systematic reviews and Meta-Analyses (S1 Fig). Four databases were searched for the review: Web of Science, PubMed, The British Library, and Google Scholar. All languages and document types were included in the searches, which took place between 7 – 9th April 2017. Given the nature of the topic, many old papers dating from the early 20th century are relevant to this review. However, the oldest paper found in the first three databases dated from 1930. Therefore, Google Scholar was searched from inception to 1950 to ensure that all documentation was included since the discovery of the full disease cycle in 1908. The search dates and results are summarized in Table 1.

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Table 1. Summary of databases, search limits and paper counts.

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The databases were searched for any combination of common schistosomiasis and water treatment terms in the title. A full list of search terms and protocol can be found in S1 Supporting Information.

Classification criteria

The first and second authors independently classified the papers and extracted relevant information. A classification system that assigns a code to each paper was developed to reduce bias in the classification process (S1 Table). First, duplicates were removed. Then, titles were classified and papers were excluded when titles indicated that they were not about water treatment or cercariae (such as code 4 –this paper is about diagnostic tests). Abstracts of the remaining papers were read, and excluded abstracts were classified (such as code 11 –this paper is primarily about shedding cercariae, not the survival of cercariae). Finally, the remaining papers were read in full and classified. Papers with code 13–19 were used for the systematic review as these papers discuss the effect of water treatment on schistosome cercariae. All codes and papers (included and excluded) are listed in S1 Table, and S2 Fig depicts the flow diagram of the selection process. Full texts were sought from the Imperial College London Library, The Wellcome Library, and the British Library. Papers in other languages were translated by Imperial College London students, and the results discussed with the first author. The two assessors met to compare and discuss the classifications, and any discrepancies were resolved.

Data extraction

The assessors summarized the papers and extracted specific information regarding each treatment method. To ensure that the same data were extracted, a table with variables relating to each water treatment method was developed (S1 Table). The assessors reviewed each other’s summaries to ensure the correct information was captured.

Results and discussion

In total, 1088 papers were found by searching the four databases. After removing duplicates, the remaining 520 studies were classified according to the codes in S1 Table. After reading the titles and abstracts, 75 studies were classified to be read in full, however 11 studies could not be acquired. Additional papers were added to the review if they were referenced in the original papers in a way that suggested that they were about human schistosomiasis and water treatment: nine papers were added in this way. Finally, 67 papers were used for the results of this review, and the flowchart in S2 Fig summarizes the classification process.

Assessing presence, motility, infectivity, and viability of schistosome cercariae in water samples

The studies in this review evaluated the effect of water treatment processes on cercariae, however the measure of effectiveness varied significantly between papers. There were four main aspects that authors assessed–the presence, motility, infectivity, and viability of cercariae. A range of methods exist to evaluate these aspects (summarized in Table 2) with differing results.

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Table 2. Summary of methods used to assess the effectiveness of water treatment methods.

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Presence of cercariae is used to assess the effectiveness of filtration. Microscopes are used to detect cercariae in water or on a recovery filter. Cercariae may be stained with a dye prior to being filtered to aid with the detection. Some studies fixed cercariae before filtration to facilitate the counting [30, 31]. This may affect results as cercariae are highly mobile and may be able to move through pores that dead cercariae would not pass through [31]. In addition, fixatives have been found to make cercariae sticky, thereby increasing the effectiveness of the filter [31].

For water treatment methods besides physical filtration, the motility of cercariae was most commonly used to assess the effectiveness of water treatment. Cercarial movement is observed under the microscope, often with dyes to facilitate observations. Initially, cercariae in treated water slow their movement, sink, and turn opaque and fluffy. They may lose their tail before the complete cessation of movement [32]. Eventually they may disintegrate, which is a sign of mortality. Most authors used motility as a measure of death. However, it has since been found that immotile or even tailless cercariae may still be infective [33], suggesting that the death of cercariae may have been determined prematurely with the motility measure. Conversely, it is also possible that cercariae that are motile are no longer infective. Cercariae of varying human schistosome species were found to have a functional longevity (time that cercariae are alive but not infective) of 50% of their survival time [34]. This would affect experiments that run longer than the functional longevity.

Infectivity can be used as a measure for testing the cercarial ability to penetrate skin. It is crucial to note that infective cercariae may die before developing into schistosomula or schistosomes, and thereby not necessarily lead to schistosomiasis infection. The attachment of cercariae to skin is visually evaluated under the microscope, or cercariae are counted before and after exposure to the host, the difference assumed to have penetrated the skin. Animal testing on mice, rats and guinea pigs is common, but cultured cells and human skin donors have also been used. Cercariae are able to determine between different types of skin, and hence the results may differ depending on the skin used [35]. Bartlett et al. showed that cultured primary keratinocytes are an alternative to animal testing, as the attachment rate is similar to that of human skin [36]. A promising method for assessing cercarial infectivity is a biosensor, as has been developed by Webb et al. [37]. Schistosome cercariae secrete elastase when penetrating the host skin, and the biosensor communicates the detection of this elastase through a loss of color.

Only viable cercariae can complete the lifecycle, and therefore transmission is cut if cercariae are rendered non-viable. Cercariae are considered viable if they can infect the human host and develop into schistosomes. Animal testing is often used to determine if cercariae developed into worms. The number of worms and severity of infection can only be determined by perfusing animals for worm counts. Another method for testing the viability of cercariae is the fluorescein diacetate (FDA) method. Viable cercariae are stained green as they take up and retain the chemical, whereas non-viable cercariae lose the green stain due to lacking esterase activity [33]. This method was initially developed to test schistosomula viability [38], and has since been used to test cercarial viability [33].

Ultimately, cercarial infectivity and viability are of greatest interest, as this determines if cercariae can infect a human host with schistosomiasis. Immobile cercariae may still be infective if in direct contact with human skin [33]. Similarly, infective cercariae may not develop into schistosomula or schistosomes and hence are not viable [33]. However, if cercariae are not infective or not viable, schistosomiasis will not be transmitted.

The effect of water storage and water temperature on schistosome cercariae

Leiper (1915) was the first to study the longevity of schistosome cercariae and found that they could survive up to 36 hours in water [39]. A more conservative estimate was used by the Great Army Medical Services in 1919 who suggested that troops fighting in tropical areas should store water for 48 hours to render it safe [18]. More recent studies found that cercariae live between 10 and 40 hours in natural conditions where water temperature varied between 20 and 30°C [15, 40, 41], but at lower temperatures their lifespan has been shown to exceed 100 hours [32]. It is now commonly accepted that cercariae can survive up to two to three days in environmental conditions [42]. Cercariae are non-feeders and depend on internal, non-renewable glycogen levels. As a result, their glycogen content decreases exponentially post-emergence [15]. However, energy levels do not correlate directly with infectivity: Whitfield et al. demonstrated that cercariae remain highly infective for up to nine hours, despite glycogen levels decreasing continually [43]. Differences in cercarial survival may be due to water temperature and water quality.

In 1966, Frick and Hillyer tested the effect of various water media on cercariae, and found that cercariae were immobile in four hours in deionized and steam-distilled water [44]. Mecham et al. tested synthetic waters to see which could maintain cercarial life the longest [45]. Soft and distilled water had the fastest cercarial die-off, aquarium water followed, and synthetic hard water could support cercarial life for up to 50 hours (pH, temperature and dissolved oxygen were controlled). Similarly, Asch showed that cercarial lifespan decreases with the addition of sodium chloride, and increases when adding glucose [40]. Overall, these studies demonstrate that the water matrix (the components of the water) can significantly affect results.

In 1911, the impact of temperature on schistosome transmission was first described. Studies found that the disease was acquired from thermal springs in Gafsa that reached temperatures of 45°C, but not from nearby hot springs that were at least 50°C [39, 46, 47]. Since this discovery, it has been shown that the lifespan of cercariae decreases with temperature, when considering the same storage times [15, 48]. This can be due to thermal intolerance or energy depletion. At temperatures exceeding 45°C, extreme temperature causes direct cercarial mortality, known as the thermal death point. The thermal death points for different schistosome species are summarized in Table 3. The point of death was visually examined through cessation of movement. At lower temperatures (below 45°C), increased cercarial death is a consequence of energy depletion. Cercarial lifespan is dependent on glycogen reserves and the rate of utilization, which is affected by temperature [49]. As temperature rises, cercariae become more active, indicated by greater displacement [50] and hence deplete their glycogen levels faster, resulting in shorter lifespans [15].

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Table 3. Thermal death points for Schistosoma species.

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Overall, it can be concluded that the time needed to reach zero cercarial survival decreases exponentially with temperature, as shown in Fig 1. An exponential regression model was fit to the data with an R2 of 0.81, indicating a moderately good fit. Cercarial survival is expected to decrease exponentially with temperature, as the depletion of glycogen is also exponential, and the rate of biological processes often increases exponentially with temperature [51]. However, only 14 data points were found and therefore more data are needed to improve the regression model. Cercariae died within minutes at 50°C, and within hours at temperatures below 44°C, though there may be some variation between species. In 1938, Porter carried out the only detailed experiments on S. haematobium cercariae survival with temperature [52]. The limited results indicate that the species is more resistant at 45°C than S. mansoni, however more data are needed to verify these results [52]. External factors such as darkness, oxygen or pH (pH 4.6–10) did not appear to affect cercarial longevity [32, 48].

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Fig 1. Water temperatures and storage times required to reach zero cercarial survival (measured when all cercariae are immobile).

Species are listed in Table 3.

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The effect of temperature on cercarial infectivity (ability to penetrate host skin) and viability (ability to develop into schistosomes) follows a similar trend. It has been shown that significantly more cercariae die during host penetration at extreme temperatures (below 10°C and above 40°C) than at 25–27°C for S. mansoni and S. haematobium [54]. Similarly, worm recovery increased with temperature to a maximum at 24–28°C for S. mansoni, followed by a steep decrease [55, 56]. In 1964, Foster was the first to study the relationship between temperature and infectivity, and found that in environmental conditions (18–30°C) temperature did not affect skin penetration and maturation to schistosomula [57]. Similarly, Lee et al. found that attachment is more dependent on cercarial age than environmental temperature (20–30°C) [14]. The effect of temperature on the functional longevity of cercariae remains to be studied.

The effect of water filtration on schistosome cercariae

Filtration is an effective water treatment method that works by physically retaining particles while allowing water to pass. Filters can retain solid matter much smaller than the pores of the filter material due to mechanical and biological processes. Granular filters, which use sand or other granular material, are suitable for use in less developed areas as the filtering media is generally locally available and no external power input is required to pass the water through the filter.

Initial studies on sand filters found that they were unable to remove cercariae from water, and troops were advised to use Doulton candles instead [18, 39, 58]. Nonetheless, Kawata showed that cercariae can be removed from water using sand with an effective size of 0.3 mm or less, and sand depth of 1.2 m [59]. Two studies confirmed that vertical sand filters are effective, however the discrepancy with earlier studies may be due to the heavy dependence on experimental conditions [60, 61]. Primarily, the size of filters varied significantly. Kawata and Fadel tested filter depths up to 1.2 m and 1.4 m respectively with positive results [59, 61], whereas Witenberg and Yofe tested filters up to 0.75 m depth, which could explain the passing of cercariae [58]. Studies found that slow filtration (0.04–0.19 m/h) retained more cercariae than more rapid filtration (0.27–0.4 m/h), as cercariae were not washed through the filter [59, 61]. These studies also showed that conditioning of filters, which develops a biologically active Schmutzdecke (biological layer formed on the surface of a sand filter), can improve the overall efficacy of sand filters. The filtration results are summarized in Table 4.

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Table 4. Summary of experimental conditions in filtration studies.

All studies used S. mansoni cercariae.

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In vertical filters, water flows from top to bottom (as opposed to horizontal sand filters in which water moves laterally downwards from one side to the other). Benarde and Johnson tested horizontal sand filters, which are often constructed in channels filled with sand [63]. Cercariae were unable to pass horizontal sand filters with 0.35 mm effective grain size and 0.9 m bed depth. These filters seem to achieve better results than vertical sand filters, the hypothesis being that the natural movement of cercariae is vertically up and down, so using a filter where water flows horizontally helps filter out cercariae [63]. Further research is needed to confirm this hypothesis.

Generally, filters used one grain size, but a filter with layers of different sized sand may prove to be beneficial and reduce the overall depth of the filter. Pre-filters would also reduce clogging and allow the filters to be used in the field with turbid water. Further research is needed to test the effect of sand size, flow rate, filter depth, filtration volume and water quality on sand filtration as a method to completely remove cercariae from water.

Other filter materials that have been tested include diatomaceous silica, a naturally occurring rock that is often used in filtration as a powder. Jones and Brady tested three types of diatomaceous silica filters and recovered no cercariae from the effluent, although no filter material sizes were specified [64, 65]. Diatomaceous silica has small, irregular pores which helps prevent cercariae from passing through the filter.

Membrane filters have been used for cercariometry (to determine the density of cercariae in water. Micro-fabrics with pore sizes of 23–30 μm have successfully filtered out cercariae [30, 31, 66–71]. When filtering natural water, several pre-filters were used to prevent the clogging of the filter. While a slow filtration rate was shown to be beneficial in sand filtration, others found that more rapid flow rates immobilized cercariae and prevented them from moving themselves through the membrane [31]. Although experiments varied in cercaria numbers, water volume, water quality, filtration rate and time, the results are consistent.

The effect of chlorination on schistosome cercariae

Water chlorination is an effective water treatment method that kills most bacteria and viruses. Chlorine is an oxidizing agent and kills pathogens through oxidation. Chlorine disintegrates the cell wall of pathogens, rendering them unviable. In chlorination, a ‘CT’ value indicates the residual chlorine dose (C) and contact time (T) required to inactivate a pathogen, and is calculated as the product of the two variables. CT values have been determined for many waterborne pathogens (see Table 5), and can be used as a process monitoring parameter, i.e. simply ensuring that the required CT is being achieved means that constant pathogen monitoring is not required. Higher CT values indicate a higher chlorine tolerance.

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Table 5. Chlorine CT values for selected pathogens, adapted from Centers for Disease Control and Prevention (2012) [72].

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The effect of chlorine on schistosome cercariae has been researched throughout the past century, with varying conclusions. In 1920, Mahnson-Bahr and Fairley found that some cercariae (unknown percentage) were still alive after 2.5 hours in water containing 4 mg/l of chlorine [10], whereas Magath found that 0.2 mg/l after 30 minutes was sufficient to kill all cercariae [73]. More recently, the WHO stated that a chlorine residual of 1 mg/l after 30 minutes is sufficient to render cercaria-free water [74]. Table 6 summarizes some of the chlorination results found in this review. Note that the contact time is the time until inactivation of all cercariae (100% kill), which in all studies was measured by complete cessation of movement of all cercariae in the sample.

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Table 6. Chlorine concentrations and contact times required to kill all schistosome cercariae in water (measured by cessation of movement).

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The data sets of all studies have been plotted in Fig 2 to show the effect of varying chlorine doses on cercariae (a more detailed graph is shown in S3 Fig). Note that these are doses of chlorine (the chlorine added to the water), not residual chlorine concentrations in the water (accounting for the reaction of chlorine with other water constituents); the lack of reporting of the chlorine residual concentration is a major weakness of many prior studies.

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Fig 2. Lethal chlorine doses for killing 100% of schistosome cercariae.

All studies used motility as a measure of death, and recorded the time at which all cercariae were motionless. Chlorine doses are plotted here because most studies did not report the residual chlorine concentrations. Species are listed in Table 6 and shown in S3 Fig.

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It is evident that in all studies the effect of chlorine increased exponentially with chlorine dose. High doses, generally above 1.5 mg/l, killed all cercariae within minutes. The data follows a clear trend and can be fit to an exponential regression model with R2 of 0.68 (Fig 2). Data is consistent at low lethal chlorine doses, but there are discrepancies at short inactivation times. This is particularly the case for inactivation times less than 30 minutes (see S3 Fig) where the lethal chlorine dose at 3 minutes, for example, varies between 1.1 mg/l and 5 mg/l. This is enforced by the variation of calculated CT values in Table 6, which vary from 3 to 30 mg-min/l. The variation may be due to differing experimental conditions, such as pH, temperature, water quality (e.g. organic matter which will react with chlorine), the form of chlorine used, chlorine measurement techniques, and schistosome species, or because of incomplete mixing of the chlorine into the water at very short contact times.

The type of water and water quality used in the experiments varied greatly. This is noteworthy, as water temperature and pH significantly affect the CT value. When sodium hypochlorite is added to water, it reacts to form hypochlorous acid. It may react further, especially under alkaline conditions, to form hypochlorite ions. The latter is a much weaker disinfectant and as a result, more chlorine is needed at higher pH to achieve the desired level of disinfection. Generally, a higher CT value is needed at lower temperature and higher pH. Lower temperature results in lower rates of chemical disinfection. In 1942, Braune tested ways to increase the efficiency of chlorine against cercariae [77]. He found that the addition of citric acid heavily accelerated the chlorine disinfection process, and suggested adding 100 mg/l of citric acid to the water. Frick and Hillyer followed in 1966 with a detailed study on the effect of chlorination on cercariae with respect to water temperature and pH [44]. They found that pH had a much greater effect on chlorine efficiency than temperature; in tap water at 20°C, S. mansoni cercariae were inactivated in 30 minutes with chlorine concentrations of 0.3 mg/l at pH 5, 0.6 mg/l at pH 7.5, and 5 mg/l at pH 10.

The chlorine demand is the difference between the amount of chlorine added to water and the residual chlorine after a given contact time. Generally, turbid water has a higher chlorine demand and consequently the CT value may be expected to increase with turbidity. Pathogens may attach to particulate matter in the water, which may encapsulate them. For this reason, CT values are usually calculated for low turbidity water, and values are then doubled in the field to account for the variability in water quality [72]. Experimental studies should account for the chlorine demand of the waters that they are testing and not simply report the chlorine dose added to the water. It is critical to measure the residual chlorine levels as chlorine decomposes rapidly, and when added to water reacts with chemicals, organic matter, and pathogens. These reactions remove some of the chlorine, hence the residual chlorine is lower than the chlorine dose added. Water that is highly contaminated therefore requires more chlorine to inactivate pathogens, to overcome the chlorine demand of the water matrix. Some authors did reference the available chlorine residual, but the time of measurement did not always coincide with the inactivation time [76, 77].

The form of chlorine used in experiments included chloramine, chlorine gas, bleaching powder, and calcium hypochlorite. These may have differing cercaricidal effects, even when used in equal available chlorine doses. Authors have reported that chloramine was less effective than chlorine, but more powerful than gaseous chlorine, and that the method of preparing chlorine also affected the results [58, 76]. Similarly, the stability of chloramine varied depending on how it was prepared. The Great Britain Army Medical Services advised against using water chlorination as a means to disinfect cercaria-infected water because chlorinated lime degraded too quickly, and the presence of sufficient free chlorine could not be ensured [18]. In addition, the method for measuring residual chlorine may be a source of error, as the DPD titration, orthotolidine method, and colorimetric test kits used in the studies have varying accuracies [79].

The species appear to impact results. S. haematobium has been found to be more sensitive to chloramine, and S. mansoni more sensitive to gaseous chlorine [58, 76]. Out of the 12 chlorine studies identified in this systematic review, only three experimented with S. haematobium or S. japonicum cercariae, stressing the need to research these Schistosoma species. In addition, it was only these three studies that simultaneously tested two species [58, 65, 76]. They found that S. haematobium and S. mansoni had differing sensitivities to chlorine, which highlights the need to conduct further research on all species under a variety of water matrix conditions.

The age of the cercariae may also play an important role in chlorination, but no research has evaluated this. Generally, studies used freshly hatched (<1 hour) cercariae. Sproule stated that fresh cercariae were more resistant to chlorine than cercariae older than 1 hour [80]. This could be due to cercariae hatching within mucus from the snail [43], which may act as a protective layer.

All authors used motility as a measure of death, and the points on Fig 2 show the time until the last cercaria stopped moving. As previously mentioned, cessation of movement does not necessarily indicate death, so the values in Fig 2 may be an underestimation of lethal chlorine doses.

In water disinfection studies, log-inactivation is commonly used to indicate what percentage of pathogens has been inactivated. As the lethal chlorine doses were recorded in previous studies when all cercariae were dead, the experiments reported a 100% kill. However, due to the small cercaria samples used (i.e. 100 or fewer cercariae), the highest log-kill that could be claimed was a 2-log kill (99% kill). Samples exceeding 1000 cercariae would need to be tested to claim a 3-log inactivation (99.9% kill), which may be challenging if evaluating the motility. Also, survival curves for cercariae follow a reverse-sigmoid form [15], which suggests that if a small sample is used, the results may be less reliable. This could explain the differences between Witenberg and Yofe, and Frick and Hillyer, as the former tested up to 200 cercariae [44], whereas the latter used as little 20 cercariae in experiments [58].

The effect of ultraviolet disinfection on schistosome cercariae

UV disinfection uses radiation in the UV wavelength range to inactivate microorganisms. This range is often referred to as germicidal, and spans wavelengths of 200–300 nm. UV is absorbed by the DNA and RNA of microorganisms, damaging cell membranes and thereby inhibiting reproduction. The sensitivity of microorganisms to UV is determined by the UV fluence (mJ/cm2)–the product of the fluence rate and exposure time [81].

In this review, two types of UV studies were found: immunization studies and water treatment studies. Immunization studies research the possibility of using UV-attenuated cercariae to develop a vaccine against schistosomiasis. The process is as follows: cercariae are irradiated with UV to a level that damages them but that does not prevent host penetration. Irradiated cercariae infect the host by penetrating the skin, but due to the UV damage are unable to develop into adult worms. Nonetheless, antibody production is triggered, and as a result, the host builds up immunity. Lighter, succeeding infections is proof that partial resistance is induced [82, 83]. The other type of UV studies found in this review was water treatment studies. These aim at using UV to disinfect cercaria-infested water and kill or inactivate cercariae. Both types of studies therefore use UV to damage cercariae, their protocol is however very different. Immunization studies irradiate cercariae so that their movement is not impaired, and they are still able to penetrate the host, whereas water treatment studies generally use higher UV fluences to instantly kill cercariae.

The effect of UV on cercariae and worm burden is summarized in Table 7. Cercariae were irradiated with fluences between 3 mJ/cm2 and 1890 mJ/cm2, all at 254nm, but the direct effect (motility, infectivity) on cercariae was often not evaluated as the focus was on worm burden (viability). Krakower was the first to research the effect of UV radiation on schistosome cercariae and found that cercariae appeared physically injured after 30 minutes, and motionless after 60 minutes exposure to sunlight when kept in a shallow container [32]. UV fluences as low as 3 mJ/cm2 have been shown to damage cercariae, slow their movement and reduce their survival [84, 85]. Higher fluences were tested in a series of experiments aimed to identify the UV fluence that triggers the highest level of host immunity [84]. The authors tested fluences up to 60 mJ/cm2 and found that 54 mJ/cm2 instantly reduced cercarial motility and damaged their physiology by inhibiting glycoprotein synthesis. A further increase to 60 mJ/cm2 made cercariae immotile and not infective. It was determined that the appropriate UV fluence for immunization studies was 18–24 mJ/cm2, as it does not inhibit cercarial penetration but results in significantly lower worm burden, as confirmed by numerous other studies [86–88]. In addition, the migration potential of adult worms is reduced, hampering their ability to migrate from skin to organs [84, 89]. Overall, cercarial movement, infectivity, and viability decreased with UV fluence.

[Figure omitted. See PDF.]

Table 7. Effect of UV disinfection on cercariae and worms.

*All fluences at 254nm.

https://doi.org/10.1371/journal.pntd.0006364.t007

The study by Ghandour and Webbe clearly demonstrates the importance of evaluating the different aspects of cercariae (motility, infectivity, viability) [82]. Irradiation for less than 0.3 minutes showed no effect on cercarial movement. However, the impact was visible when cercariae were exposed to skin; increased mortality rates during skin penetration resulted in reduced cercarial infectivity. Furthermore, the authors studied cercarial viability by examining schistosomula migration and worm burden. Most schistosomula died within days and did not develop into schistosomes, and were thus not viable [82, 95]. Similarly, Ariyo and Oyerinde showed that the damaging effect of radiation may not be immediately apparent at low UV fluences [85]. Within the first hour post-radiation, there was no effect on cercarial movement, and the attachment rate ranged between 93% and 100%, similar to the control. Nonetheless, the effect of UV became apparent in the stages following host penetration. Significantly fewer cercariae developed from schistosomula into schistosomes, and those that did had reduced fecundity, indicated by the decrease in egg load and egg viability.

The increased cercarial death associated with UV irradiation may be partially due to the increased water temperature. At the time of the studies, UV lamps emitted significant amounts of heat, easily able to increase the temperature of the water within the running time of the experiment. The risk of temperature affecting results is especially high for studies that used small water samples and long irradiation times. This could explain the positive results by Dean et al. who recovered no worms at fluence 19.8 mJ/cm2 and 3 minutes [87], whereas other studies using higher UV fluences but shorter exposure times simply achieved a worm reduction [28, 88]. The heat production and changes in temperature of the water were not recorded in the studies.

It appears that S. mansoni and S. japonicum are equally sensitive to UV, which may be explained by similar distribution of cercariae in water columns; their cercariae accumulate at the water surface, whereas S. haematobium cercariae prefer to remain beneath the surface [49]. This characteristic could lead to S. haematobium cercariae being less damaged by UV radiation, as accumulating at the bottom of the water column provides a protective water layer. Only one study tested S. haematobium, and three studies simultaneously tested two species, making it difficult to compare the results of all species.

UV radiation has been shown to inhibit the migration within the host, hampering the worms’ ability to migrate from skin to organs [82, 86]. This may be due to the physical damage of worms that develop from irradiated cercariae. One study examined the physical effect of UV on S. mansoni worms, and found that worms developed from cercariae that were irradiated for 1 minute with a Mineralight lamp (unknown fluence) had lesions, torn tubercles, and lost their spikes. This resulted in sexual anomalies and sterility [96].

The experiments generally irradiated cercariae in a shallow container with tap or distilled water. Therefore, the UV fluence measured at the surface of the water was assumed to be the UV fluence reaching the cercaria surface. This, however, is not the case since suspended particles and water itself absorb UV light, reducing the depth of UV penetration. The turbidity and depth of a water sample increase the UV fluence needed to kill cercariae, and this needs to be considered in future research.

The studies demonstrate that UV radiation damages cercariae, however the lack of measurements and controlled variables makes it difficult to draw concrete conclusions. Numerous studies did not measure the UV fluence or irradiation time, as seen in Table 7. Generally, the fluence was measured at the surface of the water with a UV meter. However, occasionally the distance at which the fluence was measured did not correspond to the surface of the water, and was hence not applicable [90]. The time of taking UV measurements may also affect the accuracy of UV fluences. Some authors took UV readings instantly after turning on the lamp, whereas others allowed the bulb to warm up for 15–30 minutes before taking measurements. Cercariae were irradiated in a variety of containers which included quartz cuvettes, glass slides and well plates, which may also affect the level of UV radiation.

The density of cercariae varied greatly between experiments, and may have affected results as cercariae may shield each other in high densities. It is crucial to carry out experiments under well-defined and controlled UV fluences to truly determine the UV-sensitivity of schistosome cercariae.

Conclusion

The results of this systematic review indicate that there are many differences in assessing the effects of water treatment processes on schistosome cercariae (i.e. motility, infectivity, viability), making it difficult to directly compare the results of studies that have used different criteria of death. It is necessary to determine at what level of motility cercariae stop being infective and viable. Methods like the biosensor and FDA described in this review may prove to be useful, as they assess the viability of cercarial populations in a water sample, not of each individual cercaria. All treatment methods should be evaluated with the same criterion that ensures cercariae are not infective or viable.

The level of variability in results differs between treatment processes. Previous studies on the effect of water storage and temperature are relatively in agreement, and indicate that water quality and pH do not greatly affect these two treatment methods. Cercariae are killed when storing water for more than one to three days, depending on temperature. The results of other water treatment processes, filtration, chlorination, and UV, are much more variable because the processes are heavily affected by experimental conditions, and require further research to obtain reproducible information. It is evident that the water matrix used in experiments hugely impacts cercaria survival. Most studies conducted experiments using lab-hatched cercariae and tap or filtered water. This builds a foundation for understanding the effect of water treatment processes on cercariae. However, it is essential to also conduct experiments under real water matrix conditions. In addition, water treatment processes should be tested individually as well as in succession of each other, such as filtration followed by chlorination, as this may achieve higher levels of cercaria removal than the treatments applied in isolation.

The variability in findings is likely also due to differences in experimental protocols and discrepancies in measuring key parameters. Chlorination studies often measured different chlorine values, such as chlorine dose, free chlorine at one-minute, or free chlorine at point of cercarial death. As there is no conversion between these measurements, the studies are incomparable. UV fluence measurements often did not correspond to the fluence reaching cercariae in the water. Filtration studies varied immensely in terms of scale and running time, making it difficult to compare the results. Experiments for all treatment processes should measure the variables needed to reliably design water treatment infrastructure.

The review has highlighted the lack of water treatment studies testing S. haematobium and S. japonicum. The majority of studies (57%) tested S. mansoni, 15% S. japonicum, and 3% tested S. haematobium. Only 12% of the studies tested more than one species (either S. mansoni and S. haematobium, or S. mansoni and S. japonicum), making it difficult to draw conclusions regarding their relative resistance to water treatment processes. The remaining 13% did not indicate which species were being tested, primarily older studies.

This review focused on the three main human Schistosoma species and no studies about water treatment processes and animal reservoir hosts were found in this review. The only studies involving animals were immunization trials using UV-attenuated cercariae. Animal reservoirs, including domestic livestock, can play a critical role in schistosomiasis transmission, especially of S. japonicum [28, 29]. Using treated water for livestock water use may contribute to lower infection rates in both humans and animals, and is therefore of medical and veterinary importance.

Implementing water treatment infrastructure could remove household water activities such as laundry, washing and recreational swimming from transmission sites. Depending on the level of treatment required to remove cercariae, other water-borne pathogens may also be inactivated, rendering water safe for drinking (e.g. if chlorination at a sufficient contact time is used). Successfully implementing and sustaining the infrastructure will require careful consideration of issues of equity of access and affordability, and it is crucial that educational programs run alongside the implementation to ensure understanding of the operation and maintenance needs. All five reviewed water treatment processes can be implemented as low-cost household or community-scale water treatment systems, yet the cost is dependent on local conditions such as availability of materials or supply chains. It is important to note that the provision of water treatment infrastructure will not remove occupational water exposure, such as fishing, from transmission sites. Nonetheless, an overall reduction in the population’s exposure to contaminated water may result in lower odds of infection and reinfection following chemotherapy, a relationship that needs to be more carefully studied and quantified. Safe water infrastructure may accelerate progress towards control and elimination of schistosomiasis by being incorporated into PC-based strategies, where it would serve to reduce the need for contact with infested water. We propose the following research priorities:

1. Determine a sensitive and specific measure for cercarial viability or infectivity.

2. Determine the effectiveness of water treatment processes against schistosome cercariae under lab and field conditions, as well as in combination.

3. Develop water treatment guidelines based on the results from water treatment experiments.

4. Examine the non-technical aspects of implementing water treatment infrastructure in a schistosomiasis-endemic region, such as cost, operation, maintenance, ownership and education.

5. Design and develop sustainable water treatment infrastructure based on the guidelines (3) and the social and economic factors (4). This may be on household scale (e.g. household sand filter) or community level (e.g. community storage tank).

6. Develop educational programs to run alongside the implementation of water treatment.

7. Incorporate water treatment infrastructure implementation and water contact awareness campaigns into schistosomiasis elimination programs.

8. Quantify the impact of water treatment infrastructure on human water contact and intensity of infection and reinfection following chemotherapy.

The literature demonstrates that all the reviewed water treatment methods have the potential to effectively remove or kill cercariae and thereby produce safe water. However, the database of required water treatment is insufficient to be able to devise guidelines for the design of water infrastructure for schistosomiasis-endemic regions. As countries target the control and elimination of schistosomiasis, it will be crucial to develop such water treatment guidelines, and to better link activities of the WASH sector to PC-based control programs.

Supporting information

S1 Fig. PRISMA checklist.

https://doi.org/10.1371/journal.pntd.0006364.s001

(DOC)

S2 Fig. Flow diagram.

Flow chart outlining the selection process for studies in this review.

https://doi.org/10.1371/journal.pntd.0006364.s002

(PDF)

S3 Fig. Lethal chlorination doses and contact times.

Data found in the systematic review, showing chlorine doses (up to 6 mg/l) and respective contact time (up to 30 minutes) required to kill cercariae. All studies used motility as a measure of death, and recorded the time when 100% of cercariae were immobile.

https://doi.org/10.1371/journal.pntd.0006364.s003

(TIF)

S1 Supporting Information. Search protocol.

Search terms and examples of search strategy for two databases.

https://doi.org/10.1371/journal.pntd.0006364.s004

(DOCX)

S1 Table. Classified included and excluded studies.

Classification codes, and a list of included and excluded studies.

https://doi.org/10.1371/journal.pntd.0006364.s005

(XLSX)

Acknowledgments

We are thankful to the librarians at Imperial College London, the British Library, and the Wellcome Library for their help in obtaining many of the studies used in this review. We are also grateful to Liliane Auwerter for translating papers written in Portuguese.

Citation: Braun L, Grimes JET, Templeton MR (2018) The effectiveness of water treatment processes against schistosome cercariae: A systematic review. PLoS Negl Trop Dis 12(4): e0006364. https://doi.org/10.1371/journal.pntd.0006364

References

1. Hotez PJ, Alvarado M, Basáñez M-G, Bolliger I, Bourne R, Boussinesq M, et al. The Global Burden of Disease Study 2010: Interpretation and implications for the neglected tropical diseases. PLoS Negl Trop Dis. 2014;8(7):e2865. pmid:25058013

2. Murray CJL, Vos T, Lozano R, Naghavi M, Flaxman AD, Michaud C, et al. Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21 regions, 1990–2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet (London, England). 2012;380(9859):2197–223.

3. Ross AGP, Chau TN, Inobaya MT, Olveda RM, Li Y, Harn DA. A new global strategy for the elimination of schistosomiasis. Int J Infect Dis. 2017;54:130–7. pmid:27939558

4. Rollinson D, Knopp S, Levitz S, Stothard JR, Tchuem Tchuente LA, Garba A, et al. Time to set the agenda for schistosomiasis elimination. Acta Trop. 2013;128(2):423–40. pmid:22580511

5. Lo NC, Addiss DG, Hotez PJ, King CH, Stothard JR, Evans DS, et al. A call to strengthen the global strategy against schistosomiasis and soil-transmitted helminthiasis: the time is now. Lancet Infect Dis. 2017;17(2):e64–e9. pmid:27914852

6. Utzinger J, Bergquist R, Shu-Hua X, Singer BH, Tanner M. Sustainable schistosomiasis control-the way forward. Lancet (London, England). 2003;362(9399):1932–4.

7. Utzinger J, Raso G, Brooker S, De Savigny D, Tanner M, ØRnbjerg N, et al. Schistosomiasis and neglected tropical diseases: towards integrated and sustainable control and a word of caution. Parasitology. 2009;136(13):1859–74. pmid:19906318

8. Sturrock RF. Schistosomiasis epidemiology and control: how did we get here and where should we go? Mem Inst Oswaldo Cruz. 2001;96 17–27. pmid:11586422

9. Sturrock RF. The schistosomes and their immediate hosts. In: Mahmoud AA, editor. Schistosomiasis. London: Imperial College Press; 2001.

10. Manson-Bahr P, Fairley NH. Observations on bilharziasis amongst the egyption expeditionary force. Parasitology. 1920;12(1).

11. Jordan P, Webbe G, Sturrock RF. Human schistosomiasis. Wallingford: CAB international 1993.

12. Ayad N. A short review of the epidemiology of schistosomiasis in Africa. Egypt J Bilharz. 1974;1:9–27. pmid:4619886

13. Haas W. The attachment response of the cercaria of Schistosoma mansoni. Influence of natural substrates and temperature. Zeitschrift fur Parasitenkunde (Berlin, Germany). 1976;49(1):63–72.

14. Lee VST, Burgess JL, Sterling CR, Lutz EA. Schistosoma mansoni: Assessment of effects of oleic acid, cercarial age and water temperature on parasite-host attraction. Int J Parasitol. 2013;43(10):837–42. pmid:23770037

15. Lawson JR, Wilson RA. The survival of the cercariae of Schistosoma mansoni in relation to water temperature and glycogen utilization. Parasitology. 1980;81(2):337–48. pmid:7443297

16. Bergquist R, Kloos H, Adugna A. Schistosomiasis: Paleopathological perspectives and historical notes. In: Jamieson B, editor. Schistosoma: Biology, Pathology and Control: CRC Press; 2016.

17. Sandbach FR. The history of schistosomiasis research and policy for its control. Med Hist. 1976;20(3):259–75. pmid:792584

18. Great Britain Army Medical Services. Memoranda on medical diseases in the tropical and sub-tropical war areas. London; 1919.

19. King CH, Bertsch D. Historical perspective: Snail control to prevent schistosomiasis. PLoS Negl Trop Dis. 2015;9(4):e0003657. pmid:25905621

20. Coelho PMZ, Caldeira RL. Critical analysis of molluscicide application in schistosomiasis control programs in Brazil. Infect Dis Poverty. 2016;5(1):57. pmid:27374126

21. Sokolow SH, Wood CL, Jones IJ, Swartz SJ, Lopez M, Hsieh MH, et al. Global assessment of schistosomiasis control over the past century shows targeting the snail intermediate host works best. PLoS Negl Trop Dis. 2016;10(7):e0004794. pmid:27441556

22. Grimes JET, Tadesse G, Mekete K, Wuletaw Y, Gebretsadik A, French MD, et al. School water, sanitation, and hygiene, soil-transmitted helminths, and schistosomes: national mapping in Ethiopia. PLoS Negl Trop Dis. 2016;10(3):e0004515. pmid:26954688

23. Erko B, Abebe F, Berhe N, Medhin G, Gebre-Michael T, Gemetchu T, et al. Control of Schistosoma mansoni by the soapberry Endod (Phytolacca dodecandra) in Wollo, northeastern Ethiopia: post-intervention prevalence. East Afr Med J. 2002;79(4):198–201. pmid:12625676

24. Jordan P, Unrau GO. Simple water supplies to reduce schistosomiasis. Trop Doct. 1978;8:13–8. pmid:628941

25. Farooq M, Nielsen J, Samaan SA, Mallah MB, Allam AA. The epidemiology of Schistosoma haematobium and S. mansoni infections in the Egypt-49 project area. 2. Prevalence of bilharziasis in relation to personal attributes and habits. Bull World Health Organ. 1966;35(3):293–318. pmid:5297627

26. Kosinski KC, Adjei MN, Bosompem KM, Crocker JJ, Durant JL, Osabutey D, et al. Effective control of Schistosoma haematobium infection in a Ghanaian community following installation of a water recreation area. PLoS Negl Trop Dis. 2012;6(7):e1709. pmid:22815999

27. Lee Y-H, Jeong HG, Kong WH, Lee S-H, Cho H-I, Nam H-S, et al. Reduction of Urogenital Schistosomiasis with an Integrated Control Project in Sudan. PLOS Neglected Tropical Diseases. 2015;9(1):e3423. pmid:25569278

28. Shi YEJ C. F.; Han J. J.; Li Y. L.; Ruppel A. Schistosoma japonicum: an ultraviolet-attenuated cercarial vaccine applicable in the field for water buffaloes. Exp Parasitol. 1990;71(1):100–6. pmid:2113005

29. Shi YEJ, C. F.; Han J. J.; Li Y. L.; Ruppel A. Immunization of pigs against infection with Schistosoma japonicum using ultraviolet-attenuated cercariae. Parasitology. 1993;106 (Pt 5):459–62.

30. Kloos H, Gardiner CH, Selim A, Higashi GI. Laboratory and field evaluation of a direct filtration technique for recovery of schistosome cercariae. The American journal of tropical medicine and hygiene. 1982;31(1):122–7. pmid:7058974

31. Prentice MA. A field-evolved differential filtration method for recovery of schistosome cercariae. Ann Trop Med Parasitol. 1984;78(2):117–27. pmid:6742925

32. Krakower CA. Some observations of the effects of physical and chemical agents on the cercariae of Schistosoma mansoni. The Puerto Rico journal of public health and tropical medicine. 1940;16(1):26–44.

33. Cheng Y, Chen X, Song W, Kong Z, Li P, Liu Y. Contribution of silver ions to the inhibition of infectivity of Schistosoma japonicum cercariae caused by silver nanoparticles. Parasitology. 2013;140(5):617. pmid:23343516

34. Thieltges DW, Rick J. Effect of temperature on emergence, survival and infectivity of cercariae of the marine trematode Renicola roscovita. Dis Aquat Organ. 2006;73(1):63–8. pmid:17240753

35. Khammo N, Bartlett A, Clothier R, Whitfield P. The attachment of Schistosoma mansoni cercariae to human skin cells. Parasitology 2001;124.

36. Bartlett A, Brown M, Marriott C, Whitfield J. The influence of human skin by schistosome cercariae: studies using Franz cells. Parasitology. 2000;121.

37. Webb AJ, Kelwick R, Doenhoff MJ, Kylilis N, MacDonald JT, Wen KY, et al. A protease-based biosensor for the detection of schistosome cercariae. Sci Rep. 2016;6:24725. pmid:27090566

38. Peak EC, I.; Hoffmann K.;. Development and validation of a quantitative, high-throughput, fluorescent-based bioassay to detect schistosoma viability. PLoS Negl Trop Dis. 2010;4(7).

39. Leiper RT. Report on the results of the bilharzia mission in egypt. J R Army Med Corps. 1915;25(1).

40. Asch HL. Effect of selected chemical agents on longevity and infectivity of Schistosoma mansoni cercariae. Exp Parasitol. 1975;38(2):208–16. pmid:240733

41. Schreiber FG, Schubert M. Experimental infection of the snail Australorbis glabratus with the trematode Schistosoma mansoni and the production of cercariae. The Journal of parasitology. 1949;35(1):91–100. pmid:18111996

42. Colley DG, Bustinduy AL, Secor WE, King CH. Human schistosomiasis. Lancet (London, England). 2014;383(9936):2253–64.

43. Whitfield PJB, A.; Khammo N.; Clothier R. H. Age-dependent survival and infectivity of Schistosoma mansoni cercariae. Parasitology. 2003;127(Pt 1):29–35. pmid:12885186

44. Frick L, Hillyer G. The influence of pH and temperature on the cercaricidal activity of chlorine. Mil Med. 1966;131(4).

45. Mecham JA, Holliman RB. Toxicity of zinc to Schistosoma mansoni cercariae in a chemically defined water medium. Hydrobiologia. 1975;46(4):391–404.

46. Conor A, Benazet L. Un nouveau foyer de bilharziose en Tunisie: La region de Kebili. Bulletin de la Societe de Pathologie. 1911;4.

47. Khalil AM. Ankylostomiasis and bilharziasis in Egypt: The control of bilharziasis in Egypt. Cairo: Department of Public Health Egypt, Interior Mot; 1924.

48. Jones MF, Brady FJ. Survival of Schistosoma japonicum cercariae at various temperatures in several types of water. Bulletin National Institutes of Health (US). 1947(189):131–6.

49. Jordan P, Webbe G. Human schistosomiasis. London: Heinemann Medical Books; 1969.

50. Valle CP J.; Gazzinelli G. Influence of temperature on the backward propulsion speed of Schistosoma mansoni cercariae. The Journal of parasitology. 1974;60(2):372–3. pmid:4821126

51. Precht HC, J; Hensel H;. Temperature and Life. New York, Heidelberg and Berlin: Springer-Verlag; 1973.

52. Porter A. The larval trematoda found in certain South African mollusca with special reference to schistosomiasis: South African Institute for Medical Research; 1938. 492 p.

53. Takaka S. On the thermal resistance of the cercaria of Schistosomum japonicum and also on the infection by bathing in warm water. Tokyo Medical News. 1924(2393).

54. Ghandour AM. A study of the relationship between temperature and the infectivity of Schistosoma mansoni and Schistosoma haematobium cercariae. J Helminthol. 1976;50(3):193–6. pmid:993578

55. Lwambo NJU, E. S.; Kruatrachue M.; Viyanant V. The host-parasite relationship between the Saudi Arabian Schistosoma mansoni and its intermediate and definitive hosts. 2. Effects of temperature, salinity and pH on the infection of mice by S. mansoni cercariae. The Southeast Asian journal of tropical medicine and public health. 1987;18(2):166–70. pmid:3118481

56. Purnell RE. Host-parasite relationships in schistosomiasis. 3. The effect of temperature on the survival of Schistosoma mansoni miracidia and on the survival and infectivity of Schistosoma mansoni cercariae. Ann Trop Med Parasitol. 1966;60(2):182. pmid:5962470

57. Foster R. The effect of temperature on the development of Schistosoma mansoni. The Journal of tropical medicine and hygiene. 1964;67:289–92. pmid:14259517

58. Witenberg G, Yofe J. Investigation on the purification of water with respect to schistosome cercariae. Trans R Soc Trop Med Hyg. 1938;31(5):549–70.

59. Kawata K. Slow sand filtration for cercarial control in North Cameroon village water supply. Water Sci Technol. 1982;14(6–7):491–8.

60. Wagner EG, Lanoix JN. Water supply for rural areas and small communities. Geneva: World Health Organization; 1959.

61. Fadel A. Slow sand filtration for cercariae removal in rural Egypt. Int J Environ Health Res. 1993;3(4):225–33.

62. Leiper RT. Report on the results of the bilharzia mission in Egypt part IV—Egyptian mollusca. J R Army Med Corps. 1916;27.

63. Benarde MA, Johnson B. Schistosome cercariae removal by sand filtration. J Am Water Works Assoc. 1971;63(7):449–53.

64. Jones MF, Brady FJ. The removal of the cercariae of Schistosoma mansoni from water by filtration through diatomaceous silica in a small model filter. Public health reports (Washington, DC: 1896). 1946;61(43):1538–43.

65. Jones MF, Brady FJ. Effects of water treatment processes on schistosome cercariae. Bulletin National Institutes of Health (US). 1947;2(189):109–30.

66. Blumenthal UJ, Jewsbury JM. Laboratory studies on a differential filtration technique for the recovery of schistosome cercariae from natural waters. Ann Trop Med Parasitol. 1983;77(4):441–2. pmid:6639189

67. Sandt DG. Direct filtration for recovery of Schistosoma mansoni cercariae in the field. Bull World Health Organ. 1973;48(1):27–34. pmid:4541145

68. Theron A. A differential filtration technique for the measurement of schistosome cercarial densities in standing waters. Bull World Health Organ. 1979;57(6):971–5. pmid:317025

69. Webbe GJ C. Filtration of cercariae through microfabric. Trans R Soc Trop Med Hyg. 1969;63(4):541–2. pmid:5821275

70. Yousif FR, M.; Ibrahim A.; El-Hommossany K.; Shiff C. Filtration, centrifugation and mouse exposure for the detection of schistosome cercariae in water. Journal of the Egyptian Society of Parasitology. 1996;26(1):249–60. pmid:8721246

71. Chandiwana S, Makaza D. Direct filtration technique for detecting the presence of schistosome cercariae in natural water. Zimbabwe Science News. 1985;19(11/12):137–8.

72. Centers for Disease Control and Prevention. Effect of chlorination on inactivating selected pathogen: Centers for Disease Control and Prevention; 2012 [Available from: https://www.cdc.gov/safewater/effectiveness-on-pathogens.html#modalIdString_CDCTable_0.

73. Magath TB. Lethal dose of chlorine for cercariae of Schistosoma mansoni. U S Nav Med Bull. 1942;40(1).

74. World Health Organization. WHO guidelines for drinking water quality: training pack. Geneva: WHO; 2000.

75. Blackmore HS. Preliminary note on the sterilization of bilharzia-infected water. J R Army Med Corps. 1928;51(4):262–4.

76. Griffiths-Jones E, Atkinson H, Hassan A. A comparison of the relative killing power of chlorine and chloramine on schistosome cercariae of the human type, together with a note on the relative stabilities of chlorine and chloramine. Ann Trop Med Parasitol. 1930;24(4):503–25.

77. Braune J. Ueber die Verhuetung der Bilharziose unter Feldzugsbedingunge, insbesondere ueber die Gewinnung von zerkarienfreiem Wasch- und Badewasser. Z Tropenmed Parasitol. 1942;46(16).

78. González JO, Biaggi N, Rivera L, J. The effect of chlorine on the motility and infectivity of the cercariae of Schistosoma mansoni. The Puerto Rico journal of public health and tropical medicine. 1945;20(3):357–61.

79. Murray A, Lantagne D. Accuracy, precision, usability, and cost of free chlorine residual testing methods. Journal of water and health. 2015;13(1):79–90. pmid:25719468

80. Sproule JC. Superchlorination of water in the field-effect on cercariae. J R Army Med Corps. 1939;72(6).

81. Bolton J, Cotton C. The ultraviolet disinfection handbook. 1 ed. Denver: American Water Works Association; 2008.

82. Ghandour AM, Webbe G. The effect of ultra-violet radiation on cercariae of Schistosoma mansoni and Schistosoma haematobium. J Helminthol. 1975;49(3):153–9. pmid:1184943

83. Moustafa H, Hassab-Allah S, Ali H, Rabee H, Ali R. Effect of ultra violet attenuated cercariae on antibody response in rabbits. IOSR JRME. 2014;4(1):54–2.

84. Ruppel AS, Y. E.; Moloney N. A. Schistosoma mansoni and S. japonicum: comparison of levels of ultraviolet irradiation for vaccination of mice with cercariae. Parasitology. 1990;101 Pt 1:23–6.

85. Ariyo AA, Oyerinde JP. Effect of ultraviolet radiation on survival, infectivity and maturation of Schistosoma mansoni cercariae. Int J Parasitol. 1990;20(7):893–7. pmid:2125983

86. Kumagai M, Sato H, Kamiya H. Migration kinetics of ultraviolet-attenuated Schistosoma mansoni in ICR mice. Kisechugaku Zasshi. 1992;41(4):300–5.

87. Dean DA, Murrell KD, Xu ST, Mangold BL. Immunization of mice with ultraviolet-irradiated Schistosoma mansoni cercariae: a re-evaluation. The American journal of tropical medicine and hygiene. 1983;32(4):790–3. pmid:6881427

88. Lin DT, F.; Wu H.; Gao Y.; Wu J.; Zhang D.; Ji M.; McManus D. P.; Driguez P.; Wu G. Multiple vaccinations with UV- attenuated cercariae in pig enhance protective immunity against Schistosoma japonicum infection as compared to single vaccination. Parasit Vectors. 2011;4(103):103.

89. Kamiya H, Ozaki T, Nakayama H, Inaba T. Immunizing potential of ultraviolet-attenuated cercariae of Schistosoma mansoni in rodent hosts. Parasitology research. 1993;79(4):293–6. pmid:8327451

90. Standen O, Fuller KA. Ultraviolet irradiation of the cercariae of Schistosoma mansoni. Inhibition of development to the admit stage. Trans R Soc Trop Med Hyg. 1959;53(3).

91. Tian F, Lin DD, Wu JJ, Gao YA, Zhang DH, Ji MJ, et al. Immune Events Associated with High Level Protection against Schistosoma japonicum Infection in Pigs Immunized with UV-Attenuated Cercariae. Plos One. 2010;5(10).

92. Nakamura SM H.; Araki K.; Tanaka H. Monoclonal antibodies against UV attenuated cercariae of Schistosoma japonicum. 1. Production and their effect on protective immunity. Kisechugaku Zasshi. 1990;39(6):543–50.

93. Moloney NA, Bickle QD, Webbe G. The induction of specific immunity against Schistosoma japonicum by exposure of mice to ultraviolet attenuated cercariae. Parasitology. 1985;90 (Pt 2):313.

94. Moloney NA, Webbe G, Hinchcliffe P. The induction of species-specific immunity against Schistosoma japonicum by exposure of rats to ultra-violet attenuated cercariae. Parasitology. 1987;94:49. pmid:3103047

95. Ghandour AM, Webbe G. Proceedings: The effect of ultra-violet irradiation on cercariae of Schistosoma mansoni. Trans R Soc Trop Med Hyg. 1974;68(1):10.

96. Mohamed SH. Scanning electron microscopical studies on the tegument of adult worms of Schistosoma mansoni originating from ultraviolet-irradiated and non-irradiated cercariae. J Helminthol. 1999;73(2):157–61. pmid:10431376