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A systematic study was conducted to investigate the cause of unusual number of gastroenteritis case patients recorded in Pakistan Institute of Medical Sciences (PIMS) Islamabad (time period). Of 220 individuals, 180 cases of gastroenteritis (81.8%) , recorded who visited the hospital compared with the baseline of an average 18 monthly cases. The symptoms were typical for gastroenteritis, predominantly diarrhoea (38.8%). The case-control study was also conducted. In case study, illness was significantly associated with drinking tap and poor filtered water (Which test of significance applied with p value and how are they significantly associated?). Enterococci and coliform (No itallics) strains were recovered from both gastroenteritis patients and water samples. Epidemiological and microbiological investigations confirm the prevalence of coliform and enterococci in Rawalpindi/Islamabad municipal water supply system. Bacteriological analysis of 50 water samples showed that more than 80% of filtered and tap water samples deviate the WHO standards showing coliform which were as high as 240 MPN/ml (expand acronym or list out in beginning index before using word) in former cases and above 2400 MPN/ml in the later. Enterococci (CFU) Expand abbreviation once) were counted in 76% and 81% filtered and tap water samples respectively. The presence of pathogens in water for drinking purpose is of public health significance considering the possibility of the presence of other bacteria, protozoa and enteric viruses that are implicated in gastro-intestinal water borne diseases.
Key words: gastroenteritis, drinkable water sources, coliforms, and enterococci
Coliform bacteria, including Escherichia spp.(expand species once) , Klebsiella spp., and Enterobacter spp., (No need of italicization) are considered to be an indicator of fecal contamination in feed and water, found in faces of the warm-blooded animals. E.coli are enteropathogenic bacteria associated with gastroenteritis particularly in developing countries. Exposure to fecally contaminated water does not always translate into infection. However, the higher the fecal bacterial levels in water, the higher the chances of pathogens to be present in significant numbers too. Dehydrating diarrhea is of critical importance related with poor microbial water quality, as it could lead to death within 48 hours after the initial symptoms. The risk factors associated with fecal waterborne diseases are gastroenteritis, typhoid and paratyphoid fevers, salmonellosis, cholera, meningitis, hepatitis, encephalitis, amoebic meningoencephalitis, cryptosporidiosis, giardiasis, dysentery, and amoebic dysentery (Are they risk factors or disease outcome?) (Jorge et al., 2010).
Similarly, inadequate treatment of gastroenteritis kills 5 to 8 thousand people per year (Kasper et al., 2005) and is a leading cause of death among infants and children under 5 (King et al., 2003) (where). These extreme cases are more predominant in countries (which) where overcrowding and poor sanitary conditions are the norm (Reference?).
Over large parts of the world, humans have inadequate access to potable water, and use sources contaminated with disease vectors, pathogens or unacceptable levels of dissolved chemicals. The distribution of drinking water is done through municipal water systems, and it has been observed that cross connection between drinking and sewage water pipes has lead to contamination of the ground water system. Many of the 3.5 billion people (source of data?) having access to piped water receive a poor or very poor quality of service, especially in developing countries where about 80% of the world population lives (Latest reference) . The Word Health Organization (1972-73) (latest references are available) estimates that 500 million diarrhea cases reportedly take place each year in children less than five years in Asia, Africa and Latin America . Several community wide outbreaks resulting from contamination of public water systems with coliform and enterococci have been reported (Engberg et al., 1998). In subcontinent, especially in Pakistan it has been estimated that each year, more than three million Pakistanis become infected with waterborne diseases as the quality of water supplies in many cities of Pakistan is deteriorating fast (Karachi, supply and sanitation services, 2004). The incidence of gastroenteritis, typhoid fever, bacillary dysentery and other enteric infections are common and are transmitted through contaminated water (WHO technical reports, 1972-1973). (Replace with new reference and be in continuity with above with latest WHO report/reference)
The Rawalpindi/Islamabad Metropolitan area is the third largest in Pakistan with a population of over 4.5 million inhabitants (Frantzeskakis 1995) where 81,996 cases of water related diseases included gastroenteritis were registered during 1994 (Din, 1997; Tahir, 1994). (reorder reference in a way of seniority; 94 and 97)
Pakistan Institute of Medical Sciences , Islamabad recorded a number of cases of gastroenteritis (exact numbers) (83%) just during the month of June 2010 (Time period) as compared to 60% cases normally (Comparative interval to be incorporated to declare this event as outbreak/epidemic-two standard deviations/10% unusual increase of the case patients for a particular period/year). Therefore, we conducted epidemiological, environmental, and microbiological investigations to determine the scope, the likely cause, and the contributing factors of gastroenteritis associated with the poor quality of drinkable water resources in Rawalpindi and Islamabad. The objective of this study is to determine the relationship between the water resource patterns and the prevalence of coliforms and enterococci.
Materials and methods
Case population and Epidemiological investigation
The staff of Pakistan Institute of Medical Sciences , Islamabad recorded the data of (exact numbers for sample size) who visited the hospital because of gastrointestinal symptoms including diarrhea, nausea, vomiting, and abdominal cramps who had illness onset during June 2010 (from when to when). (rule out any population movement/or enhanced surveillance activities by the public health authorities during that period) The line list included demographic data, and information about illness onset and symptoms. Of 220 case patients that contacted the PIMS, 180 cases were found positive during investigation (add case classification like probable, clinical or laboratory confirmed or linked epidemiologically to a laboratory-confirmed case). We conducted a case-control study to determine the risk factors to gastroenteritis. A case was defined as an illness with acute gastroenteritis defined as diarrhoea with at least three loose stools per 24 hours and/or vomiting and/or abdominal pain in a resident of Rawalpindi/Islamabad since (time interval in June, 2010). Participants were interviewed about symptoms, treatments, consumption of water from various sources (tap water, well water, bottled water), and consumption of poultry, eggs, and unpasteurised milk products. (Any informed written consent/formation of the team/training/suprevision). Ethical clearance/data analysis plan/test of significance to be applied, p-value, 95% Confidence Interval, soft ware used for analysis etc?)
All patients from whom fecal coliform/enterococci were isolated from their stool cultures were also included in this control study. All controls were matched to cases according to sex, year of birth, and residency in the metropolitan area.(how many controls selected and their method of recruitment; Inclusion and exclusion criteria adopted?)
Microbiological samples from patients
The paramedical staff of the hospital obtained stool samples from 105 (58%) of 180 patients who had gastrointestinal symptoms. The samples were analyzed for the presence of fecal Coliform, Enterococci, Salmonella and Shigella species by routine bacteriological methods by using the selective media and identified these species by Gram staining and biochemical tests.
Analysis of water samples
To study the risk factors and pattern of gastroenteritis, it was decided to investigate the municipal water supply system. For this investigation, the water samples were analyzed for certain physico-chemical and microbiological parameters by using standard methods.
Site selection and samples collection
Of 50 water samples, 17 samples were collected from municipality installed filtration units from various sites of Rawalpindi/Islamabad, 16 were taken from the wells and hand pumps (tap water) while remaining 17 were of bottled samples of various brands. All these samples were analyzed to investigate the offending microbes for gastroenteritis. Sites were selected randomly from areas of different use patterns. Water was collected in 1) sterile test tubes 2) autoclaved polystyrene 200ml bottles 3) disposible 50ml falcon tubes in day time. All samples were collected in duplicate. Water temperature was measured at each site using a stream thermometer. (How the samples were packed, labelled under international coding and transported to the laboratory in cold/hot chain? Any mismanagement in logistics and supply with ther result, the samples destroyed/or rendered useless as a result thereof ?) Whether rectal samples collected from the willing population or how many refused to part with sample?
The physico-chemical tests included the determination of temperature, turbidity, odor, color, pH, acidity, total hardness and chloride content using the methods of FAO (American Public Health Association, 1992; Food and Agriculture Organization, 1997).
Bacteriological characteristics were determined as described by Bezuidenhout (2002) and Jolt (1994). The media used for the bacteriological analysis of water include nutrient agar (NA), lactose broth (LB), and Eosin Methylene blue agar (EMB). All the media used were weighed out and prepared according to the manufacture's specification, with respect to the given instructions and directions. A serial dilution method was used for total viable count and the presumptive test for coliforms. The Most Probable Number technique was used for coliform enumeration. All plates were incubated at 37οC for 24-48hrs. Presumptive colonies were confirmed by gram staining and biochemical reactions as described by Jolt (1994) and each plate was given a positive or negative score. The sterility of each batch of test medium was confirmed by incubating one or two uninoculated tubes or plates along with the inoculated tests. The uninoculated tubes or plates were always examined to show no evidence of bacterial growth.
Case population and Epidemiological investigation
In our study results, out of 220 individuals, 180 case patients of gastroenteritis (81.8%) were diagnosed during the month of June 2010 who visited the Pakistan Institute of Medical Sciences Islamabad compared with the baseline of an average 18 monthly cases of gastroenteritis. Median age was 41 years (range 1-96 years), and 62% were women. (Calculate the attack rate (AR) for male and female case patients and also for sex specific AR. Any complications/case fatality rate. Divide the population [1-96 years] into sub groups with their numerators/denominators and find out the major age group affected and why-discuss under discussion head while comparing with latest studies. Describe the event in terms of time, place and person) The symptoms were typical for gastroenteritis, predominantly diarrhoea (38.8%) followed by the abdominal pain, nausea (16%) and fever (Figure 1). The case-control study questionnaire was sent to 175 cases and 250 controls. One hundred and fifty cases (response rate, 85.7%) and 190 controls (response rate, 74%) completed the questionnaire. Treatment was prescribed to 100 (55.5%) cases, and 80 (44.4%) received antibiotics. Of 180 case patients, 125 (69%) reported having drunk unboiled tap water at home or outside the home during the two weeks before onset of illness compared with 190 (76%) of 250 control subjects followed by the case patients found after using the municipality filtered water (30.5%). Drinking bottled and boiled water was all associated with decreased likelihood of illness. (Which test of significance was applied, on the basis of which the observations have been inked in case control study? Add the table showing frequency of three selected exposures-tap water, well water, bottled water among gastro-enteritis case patients and controls)
Microbiological samples from patients
Coliform and Enterococci were cultured from 45 (42.8%) of 105 stool samples submitted for examination from patients with gastroenteritis. No other pathogens were isolated. These cultures were identified by standard morphological Gram staining and biochemical methods.
Analysis of water samples
To investigate the source of gastroenteritis and study the pattern of infection in the metropolitan area, it was decided to collect water samples from various sites of both cities. For this purpose, a total of 50 samples, 17 sites of residential area were selected where filtration units have been installed by the municipal authorities, 16 household tap water samples were analyzed along with mineral water (250ml) of various brands were also included in this study (Table 1). (Methods section running into the result section_table 1) During survey, it was observed that the water filtration units installed in Rawalpindi/Islamabad area are equipped with UV disinfection system, but (No qualifier) at many filtration plants, this system was not working properly and bacterial contaminations were passing through the filtered water. Similarly, a compost heap for household wastes was also located near filtration units of both cities. A questioner filled by the citizens indicated that about 55% of the residents received tap water, 41% have access of filtered water while just 4% belongs to elite class used mineral water. The Rawal Lake and Simli dam are used as sources to supply water to cities, which were not fenced, allowing people and animal's access to the area.
As Table 2 indicated that physico-chemically mineral water samples were within set limits defined by the World Health Organization (WHO) and US Environmental Protection Agency (EPA). The 100% pH deviation and 35% slight discoloration observed in water samples taken from filtration units diverging the WHO standards while rest of the parameters were under control limits. Similarly, tap water samples showed deviation against WHO standards in following parameters pH (100%); discoloration (56%); chloride ions (12.5%) and turbidity measured by nephelometer (12.5%).
Water samples were also microbiologically investigated showing desperate results regarding filtered and taps water samples. All filtered and tap water samples contained coliform which were as high as 240 MPN/ml in former case and above 2400 MPN/ml in the later, which must be zero as defined by the WHO and EPA standards. Similarly, enterococci (CFU) were observed in 76% and 81% filtered and tap water samples respectively deviating the standards. (Objective scientific writing only) Mineral water samples S-48 to 50 (brand AbehyyatR) also showed coliform and enterococci as bacterial contaminants (Table 3).
(Figure 1, 13.8% of the case patients have bloody diarrhea. E. coli was a generic isolation in the absence of characterization of the strain involved (EPEC, ETEC serotype O157 H7). Usually, O157 H7 strains of E. coli are strongly associated with beef associated GE outbreaks. Did we serotype O157 H7 strain of E. coli using microbiological methods. Did we store frozen stools, sweeps, or isolates for serotyping)
On the basis of evidence from epidemiological and microbiological investigations, the prevalence of coliform and enterococci in Rawalpindi/Islamabad was caused by contaminated municipal water supply system. In the case study, illness was significantly associated with drinking tap and poor filtered water. Enterococci and coliform strains were recovered from both gastroenteritis patients and water samples. Isolation of the microbes from both the water and the patients, and finding an association between consumption of municipal water samples and illness in an analytical study, provides strong evidence that the issue was waterborne. (Where are the epidemiological findings/tables showing three variables of exposures for the limited matched analytical case control study for fixed outcome of gastroentertitis. Study design-case control study needs to be dealt in details; exhibiting strong association between exposure variables and outcome. Major revision needed )
Although the exact mechanism for contamination remained unknown, investigation of the water supply system suggested several contributing factors including the accumulation of contaminants near Rawal Lake, deposition of household wastes near filtration units, nonfunctional UV rods and unchanged/old filters in filtration plants. Tahir in (1989) reported that the water supply system of Islamabad and Rawalpindi is polluted. It was found that 76% samples in Islamabad and 82% samples in Rawalpindi were contaminated due to bacterial presence ). Though community wide waterborne outbreaks caused by coliform species have been reported previously, however the associated study conducted in multicultural federal capital area of Pakistan explored the filtered and ground tap water systems, showing higher water coliform number and enterococci in this study.
Response rates among both cases and controls were high, and therefore non-response bias probably did not affect the results of the case control study. Controls were from the same postal code area as cases, and mostly had the same source of household water, leading to risk of overmatching with possible underestimation of drinking water as risk factor. The case-control study questionnaire was sent to 175 cases and 250 controls. One hundred and fifty cases (response rate, 85.7%) and 190 controls (response rate, 74%) completed the questionnaire. This study highlights the coliform and enterococci as an important waterborne pathogen. Of 220 individuals, 180 cases were found with gastroenteritis, which visited the hospital compared with monthly 60% average cases of gastroenteritis. This problem caused considerable impact, and direct and indirect costs because of consultations, treatments, and loss of productivity. (There is no previous recording of the data in the study in question)
Filtered and groundwater (unlike the surface water which is usually disinfected before distribution) are regarded as safe source of drinking water, about 55% of the residents of Rawalpindi/Islamabad received tap water, 41% have access of filtered water while just 4% belongs to elite class used mineral water. More than 90% of the population uses municipality drinking water was found chemically and microbiologically contaminated. Physico-chemical analysis of tap and filtered water indicated the deviation of WHO standards. (result section has been reproduced here) Similarly all filtered and tap water samples contained coliform, which were as high as 240 MPN/ml in former cases and above 2400 MPN/ml in the later. Enterococci were counted in 76% and 81% filtered and tap water samples respectively. Discussion need major revisions keeping in line with the data presented in result section and then comparative study done with latest studies done in the field.
As it has been investigated from this study, in areas lacking in filtered and fresh water as in rural dwelling, educative programmes must be organized by researchers and government agencies to enlighten the villagers on the proper use of surface water. Similarly, the safety of these systems should clearly be improved including construction of offence around the lake, control of human activities to prevent sewage from entering water body, sterilization of water with ultraviolet light, installation of new filters or routine chlorination of the water to prevent the prevalence of bacterial pathogens and further cases of gastroenteritis in the city. (Recommednations should follow from conclusions)
Limitations of the study?? (What's about other tests of other bacteria, protozoa and enteric viruses;
In conclusion, as it is evident from this work that gastroenteritis is caused by water borne pathogens including prevalence of coliforms and enterococci which may be due to improper disposal of refuse, contamination of water by sewage, surface runoff, therefore programmes must be organized to educate the general populace on the proper disposal of refuse, treatment of sewage and the need to purify our water to make it fit for drinking because the associable organisms are of public health significance being implicated in one form of infection or the other.