Tổng quan về loài giardia lamblia trong nước uống - Trương Thị Thu Thủy

Tạp chí Khoa học - Công nghệ Thủy sản Số 2/2014 TRƯỜNG ĐẠI HỌC NHA TRANG • 229 VAÁN ÑEÀ TRAO ÑOÅI A REVIEW OF GIARDIA LAMBLIA IN DRINKING WATER TỔNG QUAN VỀ LOÀI GIARDIA LAMBLIA TRONG NƯỚC UỐNG Trương Thị Thu Thủy1 Ngày nhận bài: 06/11/2013; Ngày phản biện thông qua: 10/01/2014; Ngày duyệt đăng: 02/6/2014 SUMMARY The parasitic fl agella Giardia lamblia (also called as Giardia intestinalis or Giardia duodenalis) has emerged over the past decades as a major pathogen causing giardiasis. This species has two major forms in the life cycle: the trophozoite and the cyst, in which the cyst is the robust form and is very infectious and resistant in the environment. This causative agent has been associated with waterborne outbreaks. The infection can be spread directly from person to person by the Giardia infected person or indirectly from untreated water. Most people infections result from the use of water contaminated with Giardia cysts. Giadiasis is considered as a protozoan infection of gastroenteritis. An increasing number of waterborne Giardiasis outbreaks have been reported worldwide. This situation has become a major concern for safe drinking water for human consumption. In this review, relevant aspects of the biology and epidemiology of Giardiasis were discussed including the life cycle of Giardia, transmission and health effects of giardiasis, pathogenesis, methods for detection of Giardia in water and measure to control Giardia. Keywords: Giardia lamblia, giardiasis, waterborne, outbreak, gastroenteritis TÓM TẮT Loài trùng roi ký sinh Giardia lamblia (còn được gọi Giardia intestinalis hoặc Giardia duodenalis) đã trở thành tác nhân gây bệnh trong các thập kỷ qua gây ra bệnh giardiasis. Loài này có hai dạng chính trong vòng đời: cá thể dinh dưỡng và bào nang, trong đó bào nang là dạng bền vững, có tính lây nhiễm cao và có sức chống chịu trong môi trường. Tác nhân gây bệnh này gắn liền với sự bùng phát dịch bệnh lan truyền qua nước. Sự lây nhiễm này có thể xảy ra trực tiếp từ người sang người bởi những người nhiễm Giardia hoặc gián tiếp từ nguồn nước không được xử lý. Phần lớn các trường hợp lây nhiễm ở người xảy ra do sử dụng nước bị nhiễm bào nang của Giardia. Giardiasis được xem là bệnh gây nhiễm trùng dạ dày - ruột do nguyên sinh động vật. Số lượng dịch bệnh Giardiasis gia tăng đã được báo cáo ở nhiều nơi trên thế giới. Tình trạng này đã trở thành mối quan tâm hàng đầu về nguồn nước an toàn cho con người. Trong bài tổng quan này, các vấn đề liên quan về mặt sinh học và dịch tễ học của bệnh Giardiasis được thảo luận, bao gồm vòng đời của Giardia, sự lan truyền và các ảnh hưởng về mặt sức khỏe của bệnh giardiasis, sự phát sinh bệnh, các phương pháp phát hiện Giardia trong nước và biện pháp kiểm soát Giardia. Từ khóa: Giardia lamblia, bệnh giardiasis, sản sinh từ nước, bùng phát, viêm dạ dày - ruột I. INTRODUCTION Giardia lamblia is a fl agellated single-celled eukaryotic protozoan that belongs to the genus Giardia, order Diplomonadida. This parasite is also known as Giardia intestinalis or Giardia duodenalis. The organism is considered as the most commonly cause of waterborne outbreaks throughout the world. The species has two major processes in the life cycle including excystation and encystation. The organism is observed by the two forms: the trophozoite and the cyst, in which the cyst is robust and viable in long periods. Giardiasis is the disease caused by this species. Clinical infections by Giardia in humans are mainly associated with diarrhoea and malasoption. Most human infections result from the ingestion of contaminated water or food and can occur in both epidemic and sporadic forms. All groups of humans are susceptible to the infection, especially young children. Giardia lamblia has a large distribution. It can be found in a variety of animals and in environment. 1 ThS. Trương Thị Thu Thủy: Viện Công nghệ sinh học và Môi trường – Trường Đại học Nha Trang Tạp chí Khoa học - Công nghệ Thủy sản Số 2/2014 230 • TRƯỜNG ĐẠI HỌC NHA TRANG They are found in water such as sewage water, surface water, ground water and soil. Among these, sewage water is especially important since it may contain faeces from infected humans or animals (Olson et al., 1997). Giardiasis outbreaks have occurred in many places in the world. In the fall of 2004, there was an outbreak of Giardia lamblia in Bergen, Norway. Most giadiasis cases are aged between 20 - 40 years and few cases have been reported among children and elderly. Majority of the cases have symptoms of diarrhoea and abdominal cramps. Public water supply providing water to central areas of the city was reported as the source of the outbreak (Nygård et al., 2004). Giadiasis outbreaks have also occurred throughout the United States. An outbreak of giardiasis was identifi ed in central Florida in September 2006. 38 cases were diagnosed for the infection and most patients get diarrhoea and vomiting. The median age of those affected was four years old. Epidemiological investigations indicated that the source of the infection was a neighborhood interactive water fountain (Eisenstein et al., 2008). Another giadiasis outbreak occurred in New Hampshine, USA in 2007 that caused illness in 31 persons. Consuming tap water was signifi cantly associated with the disease. This outbreak was considered the largest community drinking water associated giardiasis in the USA in 10 years (Daly et al., 2010). According to CDC (Centers for Disease Control and Prevention), the total number of giardiasis in the USA increased during the years 2009 - 2010. The total number of reported cases of giardiasis increased slightly from 19,403 for 2009 to 19,888 for 2010. Most disease cases were reported among children aged 1-10 years at high rate compared to other age groups. The onset of illness occurred during early summer through early fall. The cause of the outbreak was reported due to the use of untreated water for drinking such as lakes, rivers, swimming pools that may be contaminated by a large number of Giardia cysts. The increasing number of Giardiasis outbreaks in the world that led to an increased focus on Giardia in drinking water. II. CONTENT 1. Life cycle of Giardia As mentioned above, the two forms of Giardia in the life cycle are trophozoites and cysts that leads to the successful existence of this parasite inside the human and animal body as well as in the outside environment. The cysts are resistant and can survive in the environment for a long period. The process of releasing trophozoites from cysts is called excystation and the process of transformation from trophozoites to cysts is called encystation. These processes are important for survival of the organism and for infection (Adam, 2001). Exystation is the active stage which occurs inside the host. In Figure 1 the life cycle of Giardia is schematically illustrated. After cysts are ingested, exystation occurs in the duodenum after exposure to the acidic gastric pH and pancreatic enzymes, chemotrypsin and trypsin. Each cyst releases two vegetative trophozoites. The two trophozoites replicate by asexual fi ssion in the crypts of the duodenum and upper jejunum. Excystation is facilitated by pancreatic proteases and inhibited by a trypsin inhibitor (Adam, 2001). Encystation occurs when trophozites transfer into cysts in the ileum, as a result of exposure to bile salts or from cholesterol starvation. Specifi c conditions that promotes encystation is a mildly alkalotic pH of 7.8 and bile salts plus fatty acids. The trophozites and the cysts are excreted in the faeces (Ortega, 2005). The both forms have different shapes. The cysts are approximately 7 - 10 µm in length, 11 – 14 µm in width and are round or oval in shape. The mature cyst contains four nuclei, axonemes and median bodies. Trophozoites have the shape of a teardrop with two distinct nuclei, four pairs of fl agella, two axonemes and two median bodies. They are 10 – 20 µm in length and 5 – 15 µm in width and able to attach to the mucosa of the intestine by a ventral sucking disks. The trophozoite stage is responsible for producing clinical disease in humans (Ortega, 2005). Figure 1. Giardia life cycle (CDC) Tạp chí Khoa học - Công nghệ Thủy sản Số 2/2014 TRƯỜNG ĐẠI HỌC NHA TRANG • 231 2. Transmission and health effects of Giardiasis disease The transmission of Giardia to humans is dependent upon the ingestion of cysts excreted in the faeces of infected persons or animals. Giardia is very contagious and the parasite can cause the disease named giardiasis. The parasite may be transferred directly or indirectly. Spreading of Giardia occurs directly from person to person by the transfer of cysts from the faeces of a Giardia infected person. The infection is easily spread in developing countries where the standards of hygiene and sanitation are very poor. Hospitals, nursing homes and childcare centers where transmission can be occurred from patients to patients or from patients to health care staff. Those who directly care for people infected with Giardia are at high risk of becoming infected. Sexual transmission of Giardia has been recognized among homosexual men (Ojonoma, 2008). Indirect transmission can happen through untreated water in the environment such as ponds, lakes, streams, swimming pools, and other recreational centers. The study of Sykora et al. (1988) showed that campers, hunters and hikers are easily infected by drinking untreated water from the above water sources which are contaminated with faeces of infected animals, humans or waste water. Indirect transmission also occurs by drinking polluted water which are contaminated with the faeces of infected humans or animals. Beverages are easily infected with Giardia cysts under poor hygiene practices. Widespread transmission of Giardia in humans and in the environment may led to the cause of waterborne giardiasis outbreaks in the world (Hunter and Thompson, 2005). Giardia infection can cause a variety of intestinal symptoms in both humans and other animals. Common symptoms reported in humans include diarrhoea, constipation, nausea, fl atulence, headache, abdominal pain, vomiting, weight loss, abdominal cramps, poor appetite and steatorrhea. The symptoms may be very severe and lead to considerable weight loss and dehydration, and usually appear seven to ten days after the ingestion of parasitic cysts (Faubert, 2000). Many studies report that Giardia infection may be symptomatic or asymptomatic. The study of Nikolić et al. (2011) about the epidemiological characteristics of asymptomatic and symptomatic human infection in Serbia reported that asymptomatic cases of Giardiasis represent a major proportion of the total cases of infection. However, high rates of Giardia infection were found in both asymptomatic and symptomatic populations in this country. Clinical studies have shown that ingestion of ten cysts is suffi cient to cause infection in humans, however, two thirds of the infected individuals remain asymptomatic (Ortega and Adam, 1997). Without treatment, Giardia cysts can be excreted in the stools for weeks or months. If symptoms develop, giardiasis can be classifi ed as either acute or chronic based on the degree of infection and clinical effects. Acute giardiasis has severe effects on health causing watery diarrhoea, fatty stools, nausea and vomiting. Generally, it lasts from 1 to 4 weeks, but it may persist for months. Severe infection may lead to malabsorption and malnutrition in children. Chronic giardiasis causes persistent disease, and it lasts for a long time causing malabsorption and dehydration that may lead to death. The fact that 2/3 of those who are infected without symptoms developing may complicate detection and prevention of disease outbreaks. Besides, the illness is also diffi cult to diagnose even in symptomatic individuals. This may lead to the capability to transmit the parasite to other susceptible hosts (Ryan, 1994, Ortega, 2005). 3. Pathogenesis and unaffected carriers G. lamblia is the only species within the genus Giardia able to infect humans. It is not wellknown why some infected individuals develop symptoms, while the majority remains asymptomatic carrying the parasite for years. Cases in which giardiasis has been confused with other diseases are not much given in the literature. However, Wolfe (1992) summarized the diseases that might generate similar symptoms as giardiasis and stated that: “Chronic diarrhoea from giardiasis must be differentiated from infections caused by Entamoeba histolytica, Dientamoeba fragilis, Cryptosporidium parvum, Isospora belli and Strongyloides sterocoralis as well as malabsorption, irritable bowel syndromes and infl ammatory bowel disease. Giardiasis may also mimic duodenal ulcer, hiathal hernia and gallbladder or pancreatic disease”. This author suggested that host factors such as diet, bowel motility and nutritional status probably are important for disease develops. Of parasitic factors, Wolfe (1992) refers to studies showing that there are different strains of G. lamblia and that some might be more virulent than others. Virulence is connected to enterotoxin production that has adverse effects on the host. Faubert (2000) also mentioned differences in virulence between different strains as a possible cause of differences in symptomatology among infected individuals. The number of cysts ingested and the age of the host might be important in determining the severity of the symptoms. Because of the potential severity of Tạp chí Khoa học - Công nghệ Thủy sản Số 2/2014 232 • TRƯỜNG ĐẠI HỌC NHA TRANG a Giardia infection and the problems connected to diagnosis, it is important that water management authorities should closely monitor water sources. This is benefi cial to detect and remove parasites before the water is supplied to the public. 4. Methods for detection of Giardia in water Different methods are used for detecting Giardia spp., depending on what stage of the parasite life cycle. The parasite is in the infective cyst stage when found in the environment (in drinking and bathing water, on food, etc.), while the reproductive trophozoite stage occurs within the host. This section focuses on the methods for detection of Giardia in water samples. Due to all species of Giardia persist as cysts when found in the environment, detection of Giardia lamblia in general is similar of other Giardia species. The following methods are mainly based on the review by Zarlenga and Trout (2004), who thoroughly review the most popular and up-to-date methods and techniques. Methods for detection of Giardia include several steps: sample collection, concentration, purifi cation and detection/identifi cation. 4.1. Sample collection For detection of Giardia in water samples, a large volume of water is collected and it depends on different detection protocols. In general, the amount of water is large then it increases the probability of detecting the parasite. 4.2. Concentration This is a continuous step after collecting samples. Water samples from the environment usually contain a lot of substances such as clay, mud, silt, humus, free-living microorganisms and other organic matter. The presence of such particles may interfere with the purifi cation and detection processes. Therefore, it is benefi cial to remove these at an early stage in the analysis. Concentration techniques can be divided into sedimentation technique, fi ltration technique and centrifugation technique. Sedimentation technique: Flocculation is introduced. In fl occulation, a chemical agent is added to a solution, which makes particles of similar composition (in this case the parasites) coalesce, and thereby settle out. The aim of the process is to add an agent that to a minimal degree cross-reacts with other particles than the parasite in the sample. The supernatant fl uid is removed from the sample, and the sediment consisting mainly of parasites can be further purifi ed. Filtration technique: The technique includes: Membrane fi ltration: This technique involves fi ltering concentrated or diluted water samples through a thin membrane fi lter facilitated by a vacuum pump. 10 to 40 litres is the usual norm for environmental samples of low turbidity. Extraction of the parasites from the fi lters can be done by mounting the fi lters on an inclined surface with subsequent rinsing with fi ltered water containing 0.01%Tween 80 (Nieminski et al. 1995). Depending on the pore size of the fi lter, organic contaminants present in the material retrieved from the fi lter. Further purifi cation is therefore necessary. Cyst recovery effi ciency may vary between fi lters of different composition, and membranes consisting of cellulose-acetate have been reported to yield the highest recoveries of Giardia cysts. Cartridge fi ltration: Cartridge fi lter consists of propylene fi bres inside a cartridge. The cartridge fi lter has the benefi t of being able to process larger water samples on higher fl ow rates than the membrane fi lters. After the water sample has been fi ltered through the cartridge, the fi lter is cut open, and the parasites are washed off the fi lter with an eluting solution. The solution washed off from the fi lter is collected and subjected to further purifi cation. Centrifugation technique: Centrifugation can be used both in the concentration and purifi cation step. 4.3. Purifi cation Since the concentration step rarely renders the sample free of contaminants such as algae, other microorganisms and organic matter, a purifi cation step is needed before identifi cation. The technique includes: Density gradient separation, Discontinuous gradient centrifugation and Immunomagnetic separation technique: Flotation is one of the two most commonly used techniques of density gradient separation. Flotation uses the difference in density between the cysts and unwanted contaminants. In order to separate these, the concentrated sample is centrifuged in a tube containing a solution of known density (Nieminski et al., 1995). Discontinuous gradient centrifugation (DGC) resembles fl otation in that the parasites are separated from other particles because of differences in density. However, instead of having only two layers forming one interface, DGC involves several layers of solutions with different densities, which allows the parasite cysts to be separated Tạp chí Khoa học - Công nghệ Thủy sản Số 2/2014 TRƯỜNG ĐẠI HỌC NHA TRANG • 233 from both heavier and lighter substances. The benefi t of this technique, as opposed to fl otation is that the cysts are separated from both heavier and lighter particles, and the sample may therefore become highly purifi ed. The Immunomagnetic separation technique utilises the same principles of pathogen detection with antibodies as the human body: Giardia-specifi c antibodies bound to magnetic particles (beads) are added to the sample, and the magnetic antibodies bind to the antigens of the parasite cell surface. Parasite-antibody complexes are subsequently removed from the solution by applying a magnetic force (Zarlenga and Trout, 2004). 4.4. Detection methods The detection of the organism is the fi nal step of the sample analysis and it includes the following techniques: Microscopy technique: Detection by microscopy is the most common technique and it has the benefi t of relatively cheap and uncomplicated compared to other detection techniques. Immunofl uorescent microscopy method is widely used for detection of parasite stainings. Staining of parasites can be done directly or indirectly. Direct staining is an analogus process to immunomagnetic separation: Giardia specifi c antibodies bound to fl uorescent dyes are added to the purifi ed sample, and these bind to the antigens of the parasite. The parasites are subsequently detected when the stained sample is illuminated with UV-light. Indirect stanining also uses the principle of antigen-antibody binding for detection of the parasites, but the technique involves two steps as opposed to the one-step process of direct stanining: Unlabelled antibodies are fi rst added to the sample and these binds to the parasites. Detection is then carrried out by adding a second fl uorescently-labelled antibody which binds to the primary antibody. Both direct and indirect techniques are widely used in the water monitoring, but indirect technique is preferred as this is less troubled with debris that cross-reacts with the antibodies. Flow cytometry: A fl ow cytometer is an apparatus designed for automatic detection of different kinds of microorganisms suspended in a solution. Cells are drawn into the equipment at a very high rate and they are detected when the cell intersects a laser beam. The organism is recorded based on light scattering properties (based on size, shape and composition). Organisms which have similar properties will cluster together in the output dataset. However, other particles present in the sample may also share properties with the target parasites and false positive recordings may occur. The collected specimens are then analyzed further, e.g. under a microscope. Molecular technique: The most commonly used molecular technique is IC-PCR (Immunocapture- Polymerase Chain Reaction). This method utilizes immunomagnetic separation to purify the parasites, and subsequently PCR is used for detection. Due to all organisms have certain parts of the genome that are unique, presence of such a fragment in a sample must indicate that the organism is present. PCR is a technique that amplifi es specifi c DNA sequence of the target organism. The components of the PCR reaction include: A DNA template that is extracted and purifi ed from the target organism, primers specifi c to the sequence of the DNA template, a thermostable DNA polymerase e.g. Tag polymerase is used to synthesise the DNA, dNTPs or Deoxynucleotide triphosphates, a buffer solution, and Magnesium chloride salt solution. The PCR reaction is carried out in the thermo cycler and usually starts with the initial heat step to activate DNA polymerase. The reaction includes the main following steps: denaturation, annealing, and extension. Detection of PCR-products is commonly carried out by two different techniques: The common technique is to run PCR-products by gel electrophoresis. PCR-products are also detected while the PCR is running with the presence of fl uorescently labelled probes (Real-time PCR). The major benefi t of IC-PCR and other molecular techniques in general is that they are highly specifi c to the target organism. At presence, a method is widely used around the world for detection of Giardia and Cryptosporidium in water is Method 1623 of the United States Environmental Protection Agency (USEPA). This method involves concentration of 10L environmental samples in a cartridge fi lter. Material captured on the fi lter is purifi ed with immunomagnetic separation, and specimens are subsequently stained with immunfl uorescent stains (fl uorescin - labelled antibodies) and analyzed with a fl uorescence microscope. 5. Measures to control Giardia Giardia cysts can be effectively removed or inactivated by applying control measures to the water source. Water treatment practices may include coagulation-fl occulation settling, fi ltration, disinfection and high voltage. Tạp chí Khoa học - Công nghệ Thủy sản Số 2/2014 234 • TRƯỜNG ĐẠI HỌC NHA TRANG Coagulation is the process of adding chemicals to the water causing particles to fl occulate. This affects all kinds of particles, including protozoan cysts (Betancourt and Rose, 2004). Depending on both water temperature and pH, the treatment will usually remove 50 - 70% of the water’s TOC (total organic carbon) (FHI, 2006). After the fl occulation process the particles need to be removed. In the guide for treatment of drinking water, the FHI describes three existing ways to do this: Lead the water through a process of sedimentation or air fl otation after the fl occulation process, afterwards it is fi ltrated. This procedure requires a lot of space, but it is quite simple to operate. Lead the water directly into a fi lter where all particles are fi ltered out. There are several different possible designs of such a fi lter. Lead the water directly into an alkaline fi lter. If this is done at a moderate speed the particles will be removed, in addition the water will be carbonated. The treatment processes mentioned above are methods of physically removing particles from the water. Along with those techniques, disinfection also plays a vital part in inactivating pathogens in water. For disinfection of water the use of chemical substances has been the most common method, but the focus on UV-radiation has increased. The purpose and effect of disinfection is to kill pathogens, and get their densities under a certain threshold. It is important to remember that the effectiveness of disinfection can be affected by water temperature and pH (Jarroll, 1988). It should also be mentioned that disinfectants, as opposed to fi ltrates, produce disinfection by-products (DBP). Chlorination is the most common way to disinfect drinking water. Since it is an oxidant it breaks down the cellular membrane of the pathogen. Several compounds of chlorine are used, e.g. sodium hypochlorite (NaOCl), chlorine gas (Cl2) and calcium hypochlorite (Ca(OCl)2). The effect of these disinfectant substances on bacteria is well documented, but Giardia is much more resistant. Leahy et al. (1987) showed that the concentration needed to inactivate Giardia muris cysts was 500 times higher than what was needed for E. coli. If such high concentrations was used it would probably affect the taste and smell of the water as DBP are formed. In addition, Giardia cyst can develop resistance against chlorine (FHI, 2006). As chlorine ozone is a strong oxidant, it converts complex natural organic substances in water to simpler and more biodegradable substances. In general it destroys the cellular membrane of the pathogens. If ozone treatment is used, it should be followed by activated carbon treatment to remove these substances. It can be used on large volumes of water and have few by-products (Betancourt and Rose, 2004). However, with bromide presence in water (>50µg/L), the forming of bromat must be taken into consideration. Bromat is considered a possible carcinogenic agent and probably genotoxic. Ultraviolet (UV) radiation can cause damage to the DNA or RNA in organisms, thus preventing them from multiplying, or preventing certain vital processes. Betancourt and Rose (2004) listed some advantages and disadvantages of UV treatment of drinking water. It inactivates protozoa effectively, and the need for contact time is relatively short. UV doses required for protozoa are several times higher than for bacteria and virus inactivation. Unlike other chemical disinfectants, UV radiation is a physical disinfectant that does not provide a residual to control pathogen proliferation and biofi lm formation. Therefore, it should be used it as a primary disinfected agent followed by a chemical secondary disinfectant to protect and maintain water quality in the distribution system. Otherwise, if the water has high turbidity, disinfection effect will be reduced as particles may provide shelter for pathogens. This indicates that highly turbid water may need fi ltration in advance of UV treatment. Johnstone and Bodger (2002) describes a device which disinfects water using AC (alternating current) high voltage. Experiments indicated that no viable Giardia cysts were detected using this method. However, this is a device manufactured for households. The capacity of this treatment in a large scale is unknown. III. CONCLUSIONS Giardia has proven to be a potential source that causes gastrointestinal disease in humans all over the world. Through evolution Giardia has developed a complex life cycle which includes a very robust stage outside the host that makes it able to spread effectively. The cyst form can survive for months in water, therefore drinking water is regarded as an important source of Giardiasis outbreaks. Virulence of pathogenesis is connected with enterotoxin production that has adverse effects on the host. Difference in virulence between different strains of Giardia also result in differences in symtomatology among infected persons. The majority of patients with giardiasis are asymtomatic but they can shed the cysts of the organism as carriers resulting in spread of Tạp chí Khoa học - Công nghệ Thủy sản Số 2/2014 TRƯỜNG ĐẠI HỌC NHA TRANG • 235 the disease. The infectious dose is low and humans can be infected with as few as 10 cysts. Giardiasis can be classifi ed as either acute or chronic based on the degree of infection and clinical effects. The most common symptoms of the infected persons include diarrhoea, nausea, vomitting and abdominal cramps that may lead to weigh loss and dehydration. Severe infection may lead to malabsorption and malnutrition in children. This shows that water management authorities should monitor drinking water sources closely and ensure that parasite-free water is supplied to the public. Methods to detect Giardia in drinking water are continually being improved, and molecular methods in particular become a powerful tool for detection of Giardia. Traditional treatments such as moderate chlorination are effective against many other pathogens, but extremely high concentrations are required to destroy Giardia cysts. Because of limited disinfection effect of moderate chlorination, and the potentially undesirable side effects such as taste and smell of high chlorine doses in drinking water, other methods should be considered. Disinfectants such as ozone and UV-radiation are effective alternatives. 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