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Water and : A Review

ARTICLE · JANUARY 2016 DOI: 10.9734/IJTDH/2016/21895

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Ozioma Forstinus Nwabor Emmanuel Nnamonu University of Nigeria University of Nigeria

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Available from: Emmanuel Nnamonu Retrieved on: 08 January 2016 International Journal of TROPICAL DISEASE & Health 12(4): 1-14, 2016, Article no.IJTDH.21895 ISSN: 2278–1005, NLM ID: 101632866

SCIENCEDOMAIN international

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Water and Waterborne Diseases: A Review

Nwabor Ozioma Forstinus 1, Nnamonu Emmanuel Ikechukwu 2*, Martins Paul Emenike 1 and Ani Ogonna Christiana 3

1Department of Microbiology, University of Nigeria, Nsukka, Nigeria. 2Department of Zoology and Environmental Biology, University of Nigeria, Nsukka, Nigeria. 3Department of Applied Biology, Ebonyi State University, Abakaliki, Nigeria.

Authors’ contributions

This review work was carried out in collaboration between all authors, although the work was masterminded by the first author. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/IJTDH/2016/21895 Editor(s): (1) Arun Kumar Nalla, College of Medicine, University of Illinois, Peoria, IL, USA. Reviewers: (1) Rafael A. Martínez-Díaz, Universidad Autonoma de Madrid, Spain. (2) Natthanej Luplertlop, Mahidol University, Bangkok, Thailand. (3) S. Thenmozhi, Periyar University, India. Complete Peer review History: http://sciencedomain.org/review-history/12474

Received 10 th September 2015 th Review Article Accepted 7 November 2015 Published 27 th November 2015

ABSTRACT

Despite numerous efforts by government at various levels and other agencies interested in water and its safety, waterborne diseases are still a major and environmental concern. The huge investment towards water research, although worth the spending, has not yielded the much expected result as waterborne diseases continue to plague developing countries with and Asia having the worse hit. The unavailability of pipe-borne water and the dependence of rural dwellers on surface waters which are often contaminated with faecal materials are undoubtedly the major causes of the rising prevalence of waterborne diseases. Water availability and poor hygienic practices amongst these rural dwellers are also of paramount concern as they play significant roles in the spread of water-washed diseases. Also, poor environmental practice which encourages the breeding of and other forms of vectors within residential areas contribute to the increasing prevalence of waterborne diseases. This review focuses on waterborne diseases, its classification and the various methods employed in the bacteriological analysis of water.

Keywords: Water; waterborne disease; bacteriological; conventional; molecular. ______

*Corresponding author: Email: [email protected];

Nwabor et al.; IJTDH, 12(4): 1-14, 2016; Article no.IJTDH.21895

1. INTRODUCTION defecation, malfunctioning sewage and septic systems, storm water drainage and urban runoff Countries throughout the world are concerned [23,24]. Fecal contamination of water is globally with the effects of unclean drinking water recognized as one of the leading causes of because water-borne diseases are a major waterborne diseases. The potential of drinking cause of morbidity and mortality [1,2]. Clean water to transport microbial to great drinking water is important for overall health and numbers of people, causing subsequent illness, plays a substantial role in infant and child health is well documented in countries at all levels of and survival [3,4,5,6]. The World Health economic development. The outbreak of Organization [7] estimated that globally, about cryptosporidiosis of 1993 in Milwaukee, 1.8 million people die from diarrheal diseases Wisconsin, in the United States provides a good annually, many of which have been linked to example. It was estimated that about 400,000 diseases acquired from the consumption of individuals suffered from gastrointestinal contaminated waters and seafood. Persons with symptoms due, in a large proportion of cases, to compromised immune systems, such as those Cryptosporidium [25]. Although subsequent with AIDS, are especially vulnerable to water- reports suggest that this may be a significant borne , including those infections that overestimation [26]. More recent outbreaks are self-limiting and typically not threatening to involving Escherichia coli O157:H7, the most healthy individuals [8,9]. Throughout the less serious of which occurred in Walkerton, Ontario developed part of the world, the proportion of Canada in the spring of 2000, resulted in six households that use unclean drinking water and over 2,300 cases. The number of source has declined, but it is extremely unlikely outbreaks reported throughout the world that all households will have a clean drinking demonstrates that transmission of pathogens by water source in the foreseeable future [10]. drinking water remains a significant cause of UNICEF [11] reports that 884 million people in illness. In Nigeria, cases of water related the world use unimproved drinking water source, diseases abound. Agents of these diseases have and estimates that in 2015, 672 million people been found to cut across various classes of will still use an unimproved drinking water organisms. However, most of these cases are source. In another report, UNDESA [12] put the not documented since majority of the affected worldwide estimate for people without access to individual subscribes to self-medication rather safe water at nearly 900 million. According to than seek professional medical attention. The WHO/UNICEF [13], about 2.6 billion, almost half most common waterborne diseases in Nigeria the population of the developing world, do not include Cholera, , Hepatitis, and have access to adequate . Over 80 per Typhoid [27]. Cases of water borne diseases cent of people with unimproved drinking water linked to contaminations of drinking water with and 70 per cent of people without improved pathogens have also been reported in several sanitation live in rural areas [14]. In Nigeria, a towns [28,29,30]. Waterborne outbreaks of vast majority of people living along the course of enteric disease occurs either when public water bodies still source and drink from rivers, drinking water supplies were not adequately streams and other water bodies irrespective of treated after contamination with surface water or the state of these water bodies without any form when surface waters contaminated with enteric of treatment. These natural waters contain a pathogens have been used for recreational and myriad of microbial species, many of which have or domestic purpose [31]. Instances of disease not been cultured, much less identified. The outbreak due to contaminated drinking water with number of organisms present varies considerably microbes are also reported [32,33] with the between different water types, and it is generally drinking waters sampled from Sokoto, Shuni and accepted that sewage-polluted surface waters Tambuwal towns having E. coli, Salmonella, contain greater number of bacteria than Shigella and Vibrio species far above the WHO unpolluted waters [15]. Polluted surface waters [15] allowable limit [32] and are therefore not can contain a large variety of pathogenic potable. The role of water as a vehicle for the microorganisms including viruses, bacteria and transmission of all manner of water related [16]. These pathogens, often of fecal illnesses is no longer a subject for debate, even source, might be from point sources such as ancient histories and books contain extracts municipal wastewater treatment plants indicative of this fact. Table 1 below shows some [17,18,19,20,21] and drainage from areas where of the diseases related to water and sanitation livestock are handled [22] or from non-point which are in sub Saharan Africa as well sources such as domestic and wild animal as their route of .

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Table 1. Diseases related to water and sanitation endemic in Sub-Saharan Africa

Grou p Disease Route leaving host Route of infection Disease which are Cholera Faeces Oral often water-borne Typhoid Faeces/urine Oral Infectious hepatitis Faeces Oral Giardiasis Faeces Oral Amoebiasis Faeces Oral Dracunculiasis Cutaneous Oral Diseases which are Bacillary dysentery Faeces Oral often associated with Enteroviral Faeces Oral poor Paratyphoid Faeces Oral Pinworm (Enterobius) Anal Oral Amoebiasis Faeces Oral Cutaneous Cutaneous Skin sepsis Cutaneous Cutaneous Lice and typhus Bite Bite Trachoma Cutaneous Cutaneous Conjunctivitis Cutaneous Cutaneous Diseases which are Ascariasis Faecal Oral often related to Faecal Oral inadequate sanitation Hookworm Faecal Oral/percutaneous (Ancylostoma/Necator ) Diseases with part of Urine/faeces Percutaneous life cycle of parasite in water Diseases with vectors Dracunculiasis Cutaneous Oral passing part of their life cycle in water Adapted from Bradley, D J, London School of Hygiene and

2. CLASSIFICATION OF WATERBORNE preparation of food can be the source of food DISEASES borne disease through consumption of the same microorganisms. Most waterborne diseases are Waterborne or water related diseases characterized by diarrhoea, which involves encompass illnesses resulting from both direct excessive stooling, often resulting to dehydration and indirect exposure to water, whether by and possibly . According to the World consumption or by skin exposure during bathing Health Organization, diarrheal disease accounts or recreational water use. It includes disease due for an estimated 4.1% of the total daily global to water-associated pathogens and toxic burden of disease and is responsible for the substances. A broader definition includes illness deaths of 1.8 million people every year. Further related to water shortage or water contamination estimates suggest that 88% of that burden is during adverse climate events, such as floods attributable to unsafe water supply, sanitation and droughts, and diseases related to vectors and hygiene and is mostly concentrated on with part of their life cycle in water habitats [34]. children in developing countries [13,35,36]. Most Basically, waterborne diseases can be waterborne diseases are often transmitted via transmitted through four main routes: Water- the fecal-oral route, and this occurs when human borne route, Water-washed route, Water-based faecal material is ingested through drinking route and route or water related contaminated water or eating contaminated food route. which mainly arises from poor sewage management and improper sanitation. Faecal 3. WATER-BORNE DISEASES of drinking-water may be sporadic and the degree of faecal contamination maybe low or Waterborne diseases are those diseases that are fluctuate widely. In communities where transmitted through the direct drinking of water contamination levels are low, supplies may not contaminated with pathogenic microorganisms. carry life-threatening risks and the population Contaminated drinking water when used in the may have used the same source for time

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immemorial. However, where contamination 6. ACUTE RESPIRATORY INFECTIONS levels are high, consumers (especially the visitors, the very young, the old and those Acute respiratory infections (ARI) including suffering from immunodeficiency-related pneumonia are responsible for approximately diseases) may be at a significant risk of infection. 19% of total child deaths every year [38]. In rural African regions, faecal contamination of Evidence demonstrating that good hygiene water arises from runoffs from nearby bushes practices, especially hand-washing with soap, and forest which serve as defecation sites for can significantly reduce the transmission of ARI rural dwellers. Waterborne disease can be abounds. In view of the link between ARI and caused by protozoa, viruses, bacteria, and hygiene, it can now be considered a water- intestinal parasites. Some of the organisms washed disease [40,41,42]. remarkable for their role in the outbreak of waterborne disease include Cholera, Amoebic dysentery, Bacillary dysentery (shigellosis), 7. SKIN AND EYE DISEASES Cryptosporidiosis, Typhoid, Giardiasis, Paratyphoid, Balantidiasis, Salmonellosis, United Nations Children’s Fund 2008 posits that Campylobacter enteritis, Rotavirus diarrhoea , trachoma is the world’s leading cause of E. coli diarrhea, Hepatitis A, and preventable blindness. About 6 million people are Poliomyelitis [37]. blind due to trachoma and more than 10% of the

world’s population is at risk. Globally, the disease 4. WATER-WASHED DISEASES results in an estimated $2.9 billion in lost productivity each year [43] in the US, trachoma is Water washed or water scarce diseases are caused by the trachomatis bacteria those diseases which thrive in conditions with which inflame the eye. After years of repeated freshwater scarcity and poor sanitation. Control infections, the inside of the eyelids may be of water-washed diseases depends more on the scarred so severely that the eyelid turns inwards quantity of water than the quality [38]. Examples with eyelashes rubbing on the eyeball. Flies are of water washed diseases includes; Scabies, implicated in the transmission of trachoma, and Typhus, Yaws, Relapsing fever, Impetigo, are often seen feeding on the discharge from Trachoma, Conjunctivitis and Skin ulcers. Four infected eyes. The best control method for types of water-washed diseases are considered trachoma and conjunctivitis is improved access here: soil-transmitted helminthes, acute to water for face washing. Ringworm (tinea) is respiratory infections (ARI), skin and eye also water washed disease prevalent among diseases, and diseases caused by fleas, lice, children of school age and the aged. This mites or ticks. For all of these, washing and infectious disease affects the skin, scalp and improved personal hygiene play an important keratinized tissues and is caused by a fungus role in preventing disease transmission [38]. [38].

5. SOIL-TRANSMITTED HELMINTHS 8. WATER-BASED DISEASES Helminths are intestinal worms (nematodes) that are transmitted primarily through contact with Water-based diseases are infections caused by contaminated soil. The most prevalent helminths parasitic pathogens found in aquatic host are ascaris ( Ascaris lumbricoides ), hookworm organisms. These host organisms includes; snail, ( and Necator fish, or other aquatic animal. Humans become americanus ) and whipworm ( ). infected by ingesting the infective forms or Together, these ‘geohelminths’ currently infect through skin penetration. Examples of water about one-quarter to one-third of the world’s based diseases includes Schistosomiasis population [38]. Over 130 million children suffer (cercariae released from snail, penetrate skin), from high intensity geohelminthic infections; Dracunculiasis (larvae ingested in crustacean), helminths cause about 12,000 deaths each year Paragonimiasis (metacercariae ingested in crab [39]. These diseases can be considered water or crayfish) and Clonorchiasis (metacercariae washed. Improved hygiene and sanitation can ingested in fish). These diseases can be reduce their incidence. of prevented through avoiding contact with children is also recognized as an effective control contaminated water, or use of protective clothing measure [38]. or barrier creams.

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9. INSECT VECTOR-BASED DISEASES coliforms” refers to a large group of Gram- OR WATER RELATED DISEASES negative, rod-shaped bacteria that share several characteristics. The group includes These diseases are not directly related to thermotolerant (ferment lactose and produce gas drinking water quality. They are those diseases at 45.5°C) coliforms and bacteria of faecal origin that are caused by insect vectors which breed in as well as some bacteria that may be isolated or around water bodies. Humans become from environmental sources. Thus the presence infected by being bitten by these insect vectors. of total coliforms may or may not indicate faecal However, consideration of during contamination. In extreme cases, a high count for the design, construction and operation of surface the total coliform group may be associated with a water reservoirs and canals (for drinking water or low or even zero count for thermotolerant irrigation purposes) can reduce the potential for coliforms. Such a result would not necessarily water related disease transmission. Prevalence indicate the presence of faecal contamination. It of water related diseases are high in tropical might be caused by entry of soil or organic Africa as a result of poor environmental matter into the water or by conditions suitable for management and sanitation. Drainages are often the growth of other types of coliform. In the waterlogged, hence constituting breeding sites laboratory total coliforms are grown in or on a for these insect vectors. is one of the medium containing lactose at a temperature water related diseases endemic in 117 countries range of 35-37°C. They are provisionally with about 3.2 billion people living in risk areas all identified by the production of acid and gas from over the world [44]. The report further stated that the fermentation of lactose [48]. Unlike coliforms there are about 350 to 500 million clinical cases from environmental sources, coliforms that come of malaria worldwide each year with over 1 from faecal matter can tolerate higher million deaths. About 59% of all clinical cases temperatures. These are more closely occur in Africa, 38% in Asia, and 3% in the associated with faecal pollution than total Americas. The most common vector insects are coliforms. The most specific indicator of faecal mosquitoes and flies. contamination is E. coli , which unlike some faecal coliforms never multiplies in the aquatic -borne diseases Fly-borne diseases environment [38]. E. coli is now internationally • Malaria acknowledged as the most appropriate indicator (River-blindness) of faecal pollution. In source water, its level of • Loiasis occurrence is correlated with the inputs of fecal • pollution (human or animal) [49]. Other • Filariasis organisms used as indicators of faecal pollution of water includes: Faecal Streptococci, 10. BACTERIOLOGICAL ANALYSIS OF Enterococci, Clostridium perfringens, WATER Pseudomonas aeruginosa, Hydrogen sulphide (H 2S)-producing bacteria, coliphages and other Microbial contamination is by far the most bacteriophages. serious public health risk associated with drinking-water supplies. It is impractical to 11. CONVENTIONAL METHODS FOR analyze water for every individual , BACTERIOLOGICAL ANALYSIS OF some of which can cause disease at very low WATER doses. Instead, since most diarrhea-causing pathogens are faecal in origin, it is more practical The testing of waters for pathogens has been to analyze water for indicator species that are undertaken since waterborne diseases were first also present in faecal matter. The use of recognized. In 1884, after discovery of culture indicator organisms in the bacteriological media and microscopy, Robert Koch first isolated analysis of water has remained the mainstay of a pure culture of Vibrio , and Georg Gaffky water bacteriology. For many years, total isolated the typhoid bacillus [50], the known coliforms have been used as indicators in major causes of waterborne disease in the evaluating water quality for several water uses nineteenth century: cholera and typhoid, with respect to faecal contamination [45,46]. Not respectively. The analysis of water for the all coliforms are from faecal source. Hence, presence of coliform bacteria has for long been feacal coliforms and pathogenic forms such as carried out using two classic/conventional Escherichia coli are now used largely as methods. These are the multiple fermentation bacteriological indicators [47]. The term “total tube or most probable number technique (MPN)

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and the membrane filtration methods. In recent this method include the large number of tubes years, two alternatives: the enzyme substrate needed and the long time requirement for the full (defined substrate method) and H 2S methods, test. Accordingly, this test is most conveniently have been gaining increasing popularity [38]. applied in a laboratory setting, though the presumptive test is sometimes made with field 12. MULTIPLE TUBE FERMENTATION kits. Another disadvantage of this method (and (MTF) OR MOST PROBABLE NUMBER other MPN methods) is that the result is a TECHNIQUE (MPN) statistical approximation with fairly low precision, and as such should only be considered semi- The MPN technique has been used for the quantitative [38]. analysis of drinking-water for many years with satisfactory results. It is most suitable in the 13. MEMBRANE FILTRATION METHODS analysis of very turbid water samples or if semi- solids such as sediments or sludges are to be Until the 1950s, practical water bacteriology analysed. The procedure followed is fundamental relied almost exclusively for indicator purposes to bacteriological analyses and the test is used in on the enumeration of coliforms and E. coli many countries [48]. It is customary to report the based on the production of gas from lactose in results of the multiple fermentation tube tests for liquid media and estimation of most probable coliforms as a most probable number (MPN) numbers using the statistical approach initially index. This is an index of the number of coliform suggested [52]. In Russia and Germany bacteria that, more probably than any other however, workers attempted to culture bacteria number, would give the results shown by the on membrane filters and by 1943, Mueller in test. It is not a count of the actual number of Germany was using membrane filters in indicator bacteria present in the sample [48]. conjunction with Endo-broth for the analysis of Although this test is simple to perform, it is time- potable waters for coliforms [53]. By the 1950s, consuming, requiring 48 hours for the membrane filtration was a practical alternative to presumptive results [51]. Multiple samples of the the MPN approach although the inability to water being tested are added to a nutrient broth demonstrate gas production with membranes in sterile tubes and incubated at a particular was considered a major drawback [53]. The temperature for a fixed time (usually 24 hours). If membrane filter technique shows remarkable the water source is unprotected or contamination advantage over the MPN technique in that it is suspected, serial dilutions of the water (usually could be used to test relatively large numbers of 10, 1, and 0.1 mL) may be made. Three or five samples and yields results more rapidly than the tubes per dilution are commonly used, though multiple fermentation tube technique. However, ten tubes may be used for greater sensitivity. As this method is inappropriate for turbid waters, coliform bacteria grow, they produce acid and which can clog the membrane or prevent the gas, changing the broth colour and producing growth of target bacteria on the filter [38]. The bubbles, which are captured in a small inverted technique is hence unsuitable for natural waters tube. By counting the number of tubes showing a containing very high levels of suspended positive result, and comparing with standard material, sludges and sediments, all of which tables, a statistical estimate of the MPN of could block the filter before an adequate volume bacteria can be made, with results reported as of water has passed through. When small MPN per 100 mL. Since some non-coliform quantities of sample (for example, of sewage bacteria can also ferment lactose, this first test is effluent or of grossly polluted surface water) are called a “presumptive” test. Bacteria from a to be tested, it is necessary to dilute a portion of positive tube can be inoculated into a medium the sample in sterile diluent to ensure that there that selects more specifically for coliforms, is sufficient volume to filter across the entire leading to “confirmed” results. Finally, the test surface of the membrane. Another concern with can be “completed” by subjecting positive this method is that it may not detect stressed or samples from the confirmed test to a number of injured coliforms. It was originally designed for additional identification steps. Each of the three use in the laboratory but portable equipment is steps (presumptive, confirmed and completed) now available that permits use of the technique requires 1-2 days of incubation. Typically only in the field [48]. The membrane filter method the first two steps are performed in coliform and gives a direct count of total coliforms and faecal faecal coliform analysis, while all three phases coliforms present in a given sample of water. A are done for periodic quality control or for measured amount of water is filtered through a positive identification of E. coli . Disadvantages to membrane with a pore size of about 0.45 µm,

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which traps the bacteria on its surface. The positive result indicating contamination, but membrane is then placed on selective agar or a giving no information regarding the level of thin absorbent pad that has been saturated with contamination. P/A tests are useful for screening, a medium designed to grow or permit especially in settings where most samples are differentiation of the organisms sought [54]. The expected to give negative results (e.g. treated success of this method depends on using water) [38]. Defined substrate methods typically effective differential or selective media that will require an incubation period of 18 to 36 hours. enable easy identification of colonies. However, recent studies have shown that shorter incubation periods (<12 hours) can give good 14. DEFINED SUBSTRATE TECHNOLOGY results, and if water is highly contaminated, (DST) coliforms can be detected rapidly, without a growth phase [55]. In recent times, more rapid and simple methods for the detection of indicator bacteria in water 15. HYDROGEN SULFIDE (H 2S) TEST have long been sought. One of such methods is the DST sometimes referred to as the enzyme Some group of researchers reported a simple substrate method. Defined substrate technology method for the detection of faecal contamination (DST) is a new approach for the simultaneous in drinking water. They noted that waters detection, specific identification, and confirmation containing coliform bacteria also consistently of total coliforms and Escherichia coli in water. contained organisms producing hydrogen sulfide This test uses specific indicator nutrients: ortho- (H 2S). Since H 2S reacts rapidly with iron to form nitrophenyl-β-D-galactopyranoside (ONPG) and a black iron sulfide precipitate, the authors 4-methylumbelliferyl-β-D-glucuronide (MUG). developed an iron-rich growth medium. When Enzymes produced by these indicator organisms water samples are incubated in the medium at react with these specific indicator nutrients in the 30-37°C for 12 to18 hours, production of a black nutrient medium and generally produce a striking colour indicates contamination with H 2S colour change that is easy to identify. These producing organisms [56]. The H 2S test does not tests are more rapid than conventional methods; specifically test for standard indicator species some can produce results in 24 hours or less. such as total coliforms, faecal coliforms or E. coli . Furthermore, they are more specific than Rather, a large number of bacteria can lead to conventional tests, so confirmatory tests are H2S production (e.g. Citrobacter, Enterobacter, generally not necessary. Colilert® was the first Salmonella, Clostridium perfringens ). Most of commercial DST test to receive U.S. these are faecal in origin. However both human Environmental Protection Agency approval for and animal faeces contain H 2S-producing drinking water analysis [54]. Two of the most organisms, so the H 2S test, like the total coliform relevant enzyme tests for drinking water are: test, is not specific for human faecal Beta-galactosidase and Beta-glucuronidase. contamination. The H 2S method was reviewed in Coliform bacteria produce the beta-galactosidase a WHO report [57], which found the test to be enzyme, hence when a water sample is reasonably accurate, simple and inexpensive – incubated with the Colilert ® reagent for 24 hours, approximately one fifth the cost of standard if a coliform is present, indicator nutrient is coliform tests. More reviews of literatures have hydrolyzed by the enzyme β-galactosidase of the indicated that the test detects faecally organism, thereby releasing the indicator portion, contaminated water with about the same ortho-nitrophenyl (ONPG). The free indicator frequency and magnitude as the conventional imparts a yellow color to the solution. On the methods (MTF and MF). However, the authors other hand, over 95% of E. coli produces the caution that some conditions (in particular, the beta-glucuronidase enzyme, an additional presence of sulfate-reducing bacteria) may lead constitutive enzyme that hydrolyzes the second to false positive results. False positives are of indicator nutrient, MUG. As a result of this less public health concern than false negatives, hydrolysis, MUG is cleaved into a nutrient portion and the advantages of the method (speed, (glucuronide), which is metabolized, and an simplicity and low cost) still make the H 2S test an indicator portion, methyl umbelliferone, which attractive option. The H 2S test is not mature fluoresces under ultraviolet light. DST can easily enough to replace conventional methods, but can be used in a qualitative way to measure the play a valuable role in screening water supplies. presence (P) or absence (A) of coliforms or Testing water for faecal contamination with the E. coli (P/A test). A single sample of undiluted H2S method is certainly preferable to not testing water is incubated for the appropriate time, with a at all [38]. A better, yet faster and more accurate

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method is the molecular method such as the use of EPEC and EHEC) and bfpA (structural gene of multiplex PCR technique [58] to detect for the bilus-forming pili of typical EPEC) for toxigenic and pathogenic strains. EPEC, aggR (transcriptional activator for the AAFs of EAEC) for EAEC, elt and est for ETEC 16. MOLECULAR METHODS FOR (heat-labile and heat-stable enterotoxins of BACTERIOLOGICAL ANALYSIS OF ETEC), ipaH (invasion plasmid antigen H found WATER in EIEC and Shigella ) for EIEC, and stx (Shiga toxins 1 and 2 and variants) for STEC. The assay Although traditional/conventional microbiological was tested for specificity with reference strains methods such as MPN, MF, DST and serological and clinical isolates and was used to detect E. tests are clearly useful in the bacteriological coli in stool samples of children with and without analysis of water, methods based on specific diarrhea. They found that atypical EPEC was the nucleic acids have significant additional most commonly isolated category of advantages for the detection of indicator bacterial diarrheagenic E. coli . A multiplex technique that species. Traditional methods for the detection targeted the Vibrio cholera virulent genes hlyA, and identification of bacteria rely on growing the ctxB, tcp1 have been proposed to be a reliable organism in pure culture and identifying it by a method to detect toxigenic-pathogenic strains of combination of staining methods, biochemical V. cholera [61]. Various mPCR methods have reactions and other tests. This applies equally to also been developed to identify specific groups of detection of environmental organisms (in soil or E. coli [86]. A particularly large number of water), bacteria in food (including milk and techniques have been reported for EHEC and drinking water) or pathogens in samples from STEC E. coli , typically targeting two or more of patients with an infectious disease. However the following: genes encoding for Shiga toxins 1 these methods are slow, requiring at least 24 h and 2 (stx1 and stx2 ), intimin ( eaeA ), or several weeks for slow-growing organisms enterohemolysin ( ehxA ), a mega plasmid- such as Mycobacterium . In addition, encoded adhesion, Saa ( saa ), a subunit gene there are some bacteria such as Mycobacterium (subA ) and/or a novel toxin [63,64,65,66,67, leprae (the causative agent of ) that still 68,69,70]. Other mPCR assays have enabled cannot be grown in the laboratory. The use of identification of the E. coli serotypes O157, O26, molecular techniques which rely on the O111, O103, O121, O145 [63,69,71,72] and amplification and detection of specific nucleic E. coli O157:H7 [73,74,75,76,77,78], acids offers a fast and reliable alternative and enteroaggregative E. coli [79,80,81,82], E. coli hence, they are the choice methods. Here we safety and laboratory strain lineages (K-12, B, C, reviewed many of these techniques, the and W) [83], uropathogenic E. coli [84], and advantages of these methods, and their use in enterotoxigenic E. coli [85]. investigating waterborne E. coli . Some of these molecular techniques includes; multiplex PCR, 18. REVERSE TRANSCRIPTION PCR reverse transcription PCR, and microarrays. Although DNA based PCR methods are reliable, 17. MULTIPLEX PCR (mPCR) the inability of DNA-based molecular methods to distinguish between live and dead cells is a Multiplex PCR is a recent molecular method significant limitation toward monitoring possible used in the detection of bacteria in samples. pathogens in water samples [86]. Another major Among DNA based methods, monoplex PCR is limitation of the standard PCR methods is it imperfect because it does not give concurrent inability to detect the presence of certain viruses detection of the toxigenic-pathogenic potential involved in waterborne disease outbreaks. To and regulating factors [59]. A key advantage of address these limitations, various research have mPCR involves increased specificity because investigated the potentials of mRNA as possible more than one gene is targeted [58,104]. Further, target for detecting viable bacteria cells [87,88]. when the method is combined with real-time Early studies targeted E. coli mRNA for two PCR, target quantification is possible. A genes ( groEL and rpoH ) involved in stress particularly useful mPCR method to differentiate response, as well as a gene ( tufA ) for an between the E. coli categories was recently abundant cellular housekeeping protein, and reported. The researchers used seven primer concluded that mRNA was indeed an effective pairs to differentiate EPEC (typical and atypical), indicator of viability [89]. In order to obtain a EAEC, ETEC, EIEC, and STEC [60]. The method sensitive method for detecting a specific mRNA, involved targeting eae (structural gene for intimin a DNA copy is made initially using an

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RNA-directed DNA polymerase reverse afford while majority still rely on the available transcriptase. Following the initial reverse water sources irrespective of their physical, transcription, a standard PCR can be used to chemical and biological state. Occurrence of amplify the DNA strand produced. This method, pathogenic microorganisms in fresh water bodies reverse transcript PCR or RT-PCR, provides a demands routine assessment as a means of very sensitive method of detecting the presence forestalling future outbreaks. However, the of a specific transcript within small sections of a detection of water borne diseases and their bacterial population. A serious limitation of RT- vectors has become an evolving art which PCR as described above is that it is not easily requires some sought of professionalism. quantifiable. It can be used to detect a transcript Conventional methods such as Membrane but not (or at least not easily) to determine how filtration and the MPN requires prolonged timing much of that transcript is present [90].The and are in most cases not exact in their specificity of mRNA detection methods has been conclusions. Molecular techniques on the other further advanced by combining reverse hand though efficient and reliable requires transcription with other molecular techniques sophisticated instrumentation and expertise such as multiplex PCR (RT mPCR). For which are missing in most developing countries example, viable E. coli O157:H7 was detected where waterborne diseases still constitute a using RT mPCR targeting the lipopolysaccharide problem. Hence the scourge of waterborne gene ( rfbE ) and the H7 flagellin gene ( fliC ) [91]. disease remains unabated. There is therefore a Reverse transcription has also been combined need to build on available methods especially with real-time multiplex PCR technologies (RT fast and reliable methods which will sufficiently mRT PCR). For example, techniques have been address the crisis within the developing developed to detect mRNA encoded by rfbE and countries, not necessarily discarding the existing eae genes of E. coli O157:H7 in pure cultures methods but strengthening them to be able to and bovine feces [92]. tackle the need of the time. It is also pertinent that enlightenment campaigns aimed at 19. MICROARRAYS educating the people of the need for proper handling of water be embarked upon as a means Microarrays probably represent the future for to addressing the existing water crisis. detecting waterborne pathogens because of the tremendous investigative power provided by the CONSENT parallel detection of many genes [86]. For instance, one microarray was able to detect 189 It is not applicable. E. coli virulence and virulence-related genes as well as 30 antimicrobial resistance genes [93]. A ETHICAL APPROVAL large number of other studies have developed microarray technology for E. coli , targeting It is not applicable. virulence genes [94,93,95,96,97], O-antigen gene clusters [98], or antibiotic resistance genes COMPETING INTERESTS [99,93,96,97]. Microarray technology has also been developed to detect other bacterial Authors have declared that no competing pathogens, targeting virulence genes [100,101], interests exist. 23S rRNA gene [101], 16S rRNA gene [102], cpn60 gene (GroEL, a chaperonin protein), wecE gene (enterobacterial common antigen REFERENCES biosynthesis) [102], and gyrB gene (subunit B of bacterial gyrase) [103]. 1. Clasen T, Schmidt W, Rabie T, Roberts I, Cairncross S. Interventions to improve 20. CONCLUSION water quality for preventing diarrhoea: systematic review and meta-analysis. The importance of water to man cannot be over British Medical Journal. 2007;1-10. emphasized, however its role as vehicle in the DOI: 10.1136/bmj.39118.489931.BE transmission of pathogenic organisms has Available:http://www.bmj.com/cgi/reprint/33 become a source of concern and fear to man as 4/7597/782 he cautiously scrutinizes every drop of water 2. World Health Organization. UN-water before consumption. Unfortunately, the cost for global annual assessment of sanitation safe water is that which only a very few can and drinking-water GLAAS): Targeting

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