ANTIBIOGRAM OF DIARRHOEAGENIC ESCHERICHIA COLI AND NUTRITIONAL STATUS OF DIARRHOEIC PATIENTS UNDER FIVE YEARS ATTENDING SELECTED HOSPITALS IN KADUNA STATE, NIGERIA

ABSTRACT
Escherichia coli infections and poor nutritional status have implications on the growth and development of children under five years, physically, mentally and healthwise with consequences such as diarrhoea, stunting, wasting, underweight and often times leading to death, depending on their severity. This study evaluated the antibiogram of Escherichia coli O157 and VerocytotoxigenicEscherichia coli(VTEC)and the nutritional status of diarrhoeic children under five years in Kaduna State, Nigeria, using Conventional isolation methods, latex agglutination tests, VTEC-ELISA tests, Chi-square (SPSS Version 19) and WHO Antro (Version 3.2.2). Purposive sampling was used to select 350 children presenting with diarrhoea in six government hospitals within the three senatorial zones of Kaduna State. The results obtained revealed that 76(21.7%) of the 350 stool samples were positive for E. coli, 28(36.8%) of which were positive for E. coli O157:H7serotype and 1(1.3%) verocytotoxigenic E. coli (VTEC) serotype. High susceptibility to ciprofloxacin, chloramphenicol and high resistance to sulphamethoxazole, cefotaxime, amoxicillin, gentamicin and tetracycline by the isolates were observed. The nutritional status assessment showed 34.3, 24.3 and 13.1% of the children were stunted, wasted and underweight, respectivelyand Kaduna South senatorial zone also recorded the highest prevalence of undernutrition, indicating poor nutrition and poor health accumulated overtime. The study concluded that antibiotics have not been very effective in the treatment of E. coli-related diarrhoea, with VTEC now emerging in this part of the world, making it a serious public health issue and its severity enhanced by malnutrition, confirming that the relationship between diarrhoea and malnutrition is bidirectional, resulting in severe limitations in the growth and development of the children, some of which may be irreversible. The study  therefore  recommends  the implementation  of programmes geared towards good hygiene, good nutrition and good health.


CHAPTER ONE
1.0       INTRODUCTION
Diarrhoea can be defined as the occurrence of three or more loose, liquid or watery stools or at least one bloody loose stool in a 24h period(Gondweet al., 2006).Diarrhoea is also defined as the occurrence of loose or watery stools at least three times per day, or more frequently than normal for an individual (WHO,2013). Diarrhoeal diseases of the bowel make up a veritable augean stable of entities. Microbiological agents cause many diarrhoeal diseases; others arise in the setting of malabsorptic disorder and idiopathic inflammatory. An increase in stool mass, stool frequency or stool fluidity is perceived as diarrhoea by most patients (Crawford, 1999). For many individuals, this consists of daily stool production in excess of 250 gm, containing 70 – 95% water. More than 14 litres of fluid may be lost per day in severe cases of diarrhoea (i.e the equivalent of the circulating blood volumes). Diarrhoea is often accompanied by pain, urgency, perianal discomfort, and incontinence. Low-volume, painful, bloody diarrhoea is known as dysentery (Crawford, 1999).

The World Health Organisation defined diarrhoea as the voiding of more than two unformed watery stools in any 24 h period, or any voiding of watery stools if accompanied by fever, abdominal pain and / or vomiting (Crawford, 1999 and Gondwe et al., 2006). Diarrhoea accounts for more deaths in childhood than any other disease in the developing world(Crawford, 1999).It has been pointed out that in developing countries; a child of less than seven years of age still has a 50% chance of dying from diarrhoeal diseases (Crawford, 1999; Guerrant et al., 2001; Gondwe et al., 2006). Infantile diarrhoea remains one of the leading causes of childhood morbidity and mortality in developing countries, with children in the developing world having an average of 5-6 episodes a year (Crawford, 1999).There are three major forms of diarrhoea namely: acute watery diarrhoea, acute bloody diarrhoea and persistent diarrhoea (Guerrantet al., 2001).Diarrhoeal diseaseforms one of the two major killer diseases in children under five years of age in the developing world. Escherichia coli is one of the major bacterial causes of diarrhoeal diseases (Abdullahi et al., 2010). The bacterium E. coli is one of the best and most thoroughly studied free-living organisms. It is also a remarkably diverse species because some E. coli strains live as harmless commensals in animal intestines. External contact and subsequent ingestion of bacteria from faecal contamination can cause detrimental health effects (Money et al., 2009).

Acute watery diarrhoea is diarrhoea with a high volume of watery stool occurring over a period of less than 14 days. It usually results in severe dehydration if intervention measures are not sought. Acute bloody diarrhoea is diarrhoea manifested by loose or watery stools with the shedding of red blood cells. Persistent diarrhoea is usually associated with loose or watery stools with or without visible blood occurring over a period of 14 days. Diarrhoeal disease caused by microbial agents is principally a food borne and water borne illness. Foodborne and waterborne illnesses are leading global health problems accounting for more morbidity and mortality than tuberculosis and malaria (Besser et al., 2003).According to World health organization (WHO) Report, approximately 11 million children under the age offive, die because of E. coli-mediated gastroenteritis (WHO, 2005).

Nutrition is a science that concerns itself with the relationship between food and the functioning of the human body and it encompasses the ingestion of food, digestion, absorption, metabolism and excretion of waste products. Nutrition plays a key role in physical, mental and emotional development of children and much emphasis has been given to provide goodnutrition to growing populations especially in the formative years of life, which is one thousand (1,000) days. Globally, more thanone- third of child deaths are attributable to under nutrition. Malnutrition refers to a case of faulty nutrition which can spread through a broad spectrum, from under nutrition to over nutrition, causing diseases like Obesity, Marasmus, Kwashiorkor, Protein-Energy Malnutrition (PEM), underweight and other chronic diseases (Alim and Jahan, 2012).Diarrhoeal illness isoften attributed to contaminated water (or food) consumptionalthough the percentage specifically due to waterbornepathogens is still unknown (Hunter et al., 2003). Transmission of agents that cause diarrhoea are usually by the faecal oral route, which include the ingestion offaecal contaminated water or food, person to person contact and direct contact with infected faeces. Host factorsthat increase susceptibility to diarrhoea include under nutrition, current or recent measles and immune deficiencyor immunosupression (Andu et al., 2002). Among the bacterial pathogens of diarrhoeal diseases, the most commonly implicated in the endemic form of childhooddiarrhoea on a global scale are strains of Escherichia coli (Huilan et al., 1991; Iruka et al., 2003; WHO, 2005).Diarrhoeal diseases and other related gastrointestinal disorders are one of the most important causes of illness and death all over the world, particularly among infants and young children (Elias et al., 1999;Clarke, 2001; Sarantuya et al., 2004). The major causes of diarrhoeal illness include, among others, limited access to / or poor quality of water, poor food hygiene, and sanitation. The bacterial pathogens usually responsible for diarrhoeal illness include Escherichia coli, Shigella, Salmonella, Campylobacter, Yersinia, Aeromonas(Presterl et al., 2003).The relationship between diarrhoea and malnutrition is bidirectional: diarrhoea leads to malnutrition while malnutrition aggravates the course of diarrhoea. Many factors contribute to the detrimental effect of diarrhoea on nutrition. Reduced intake (due to anorexia, vomiting, andwithholding of food), maldigestion, malabsorption, increased nutrient losses, and the effects of the inflammatory response are some of the factors involved in malnutrition (Nel, 2010).

Diarrhoeal disease may cause, precipitate, or exacerbate protein-energy and micronutrient malnutrition through five possible mechanisms: 1) reduced food intake-reduction of food intake during diarrhoea may be due to the child's anorexia, maternal food-withholding behaviour, or both; 2) decreased absorption of nutrients due to structural damage to the intestine, as well as the physical action of increased intestinal movement and reduced fluid transit time, all interact to produce decreased absorption of nutrients; 3) increased catabolic losses-under the influence of the inflammatory process, diarrhoea of infectious origin induces an average daily negative nitrogen balance of 0.9 g/kg/day, as muscle protein is converted to glucose through gluconeogenesis by the liver; this glucose is used as a fuel by tissues to sustain the hypermetabolism associated with fever; 4) nutrient loss from the intestine-in diarrhoea nutrients are lost directly from the intestinal tract; 5) metabolic inefficiency due to micronutrient deficiency-the increased rate of tissue synthesis displayed by children recovering from protein-energy malnutrition may be hampered by a limited supply of nutrients from the body pool, which in turn may not be replenished fast enough by dietary intake (Martinez and Tomkins, 1993).

1.1 Statement of the research problem
Diarrhoeal diseases ranks second as a major cause of morbidity among the modifiable diseases in Nigeria, where, according to the Federal Statistic Bulletin, 300 children die every day from malnutrition and dehydration caused by diarrhoea (Barua, 1981; FMH, 1987).Severe and prolonged episodes of diarrhoea cause malnutrition in individual patients; while malnourished children are more likely to develop complications with diarrhoea. Many studies, addressing the relationship between diarrhoea and malnutrition, have demonstrated a detrimental effect of diarrhoea on nutritional status(Nel, 2010).

The impact of diarrhoeal illness is most felt in sub-saharan Africa where out of the 25million children born each year, 4.3 million are expected to die by the age of 5 years and about 20% of these deaths will be from diarrhoea (Cunliffe et al., 1998). Although no specific organism has consistently been identified as a cause of persistent diarrhoea, in certain geographic areas Entero-aggregative E. coli, Cryptosporidium, and Giardia lamblia have been identified as important causes of persistent diarrhoea and malnutrition( Nweze, 2010).In Nigeria, the aetiology of diarrhoeagenic bacteria and the virulence of various Escherichia coli pathotypes have not been well studied as most currently published data are from the southwestern axis of the country (Nweze, 2010). Although in developed countries and in a few developing countries, the mortality rates have declined considerably in recent times due to improvement in general hygiene and advances in health care. The problem still persists in so many other countries where outbreaks of diarrhoeal diseases continue to affect millions of infants and young children (Hart, 1989; Presterlet al., 2003).

Illnesses such as irritable bowel syndrome, Crohn‘s disease and ulcerative colitis cause the patient to exhibit similar symptoms to individuals who have developed gastroenteritis due to infection with a pathogenic organism capable of causing diarrhoeal disease. After initial diagnosis of an inflammatory bowel disease, there is typically no further investigation if a patient suffers a relapse due to the underlying cause of the symptoms being known. There is however, the potential that the onset of inflammatory bowel disease is triggered by infection with a pathogenic organism such as Escherichia coli which results in gastroenteritis. As such this is the primary cause of the symptoms rather than the result of a weakened immune response due to the inflammatory bowel disease which is often considered to be the case (Burke and Axon, 1988).

Similar effects have been seen after a bout of traveller‘s diarrhoea with patients still suffering the effects of the infection two weeks after initial contraction. In some individuals this long term effect of the traveller‘s diarrhoea results in the onset of irritable bowel syndrome, again suggesting that these pathogens are the primary cause of such illnesses (Connor, 2005).The occurrence and distribution of bacterial pathogens causing diarrhoea in humans has beenshown in various studies (Reyes et al., 2009). Bando et al. (2009) reported that a high incidence of infantile diarrhoea is associated with atypical Enteropathogenic E. coli (EPEC)

1.2 Justification
Worldwide, diarrhoeal diseases are estimated to cause over 2 million deaths annually (WHO, 2004), accounting for approximately 4.3% of the total number of deaths and as a result ranking diarrhoeal diseases as the 5th highest cause of death globally (UNICEF/WHO, 2011). Deaths due to diarrhoeal diseases are more common in third world countries with a lower socio-economic standing and which has a significant impact on child health and mortality rates (WHO, 2004).Diarrhoea is one of the leading causes of death among children under five globally. More than one in ten child deaths – about 800,000 each year – are due to diarrhoea. Today, only 44% of children with diarrhoea in low-income countries receive the recommended treatment, and limited data in literature suggests that there has been little progress since 2000 (UNICEF/WHO, 2013).

Acute diarrhoeal disease is a major public health problem throughout the world, with over two million deaths occurring each year, and affecting mostly children under 5 years of age in developing countries (Kosek et al., 2003; Bryce et al., 2005). This disease is especially common in developing countries with poor hygiene and sanitation, and with limited access to safe drinking water. Underlying conditions such as malnutrition, which increase the risk of contracting diarrhoea, are also common in these countries. These factors may result in a signficant disease burden with economic effects due to direct medical costs, loss of work, lower quality of life and mortality (Bonkongou et al., 2010; Bonkongouet al., 2013).Bacteria such as Escherichia coli, Shigella and Vibrio cholerae are all known to cause diarrhoeal diseases and are identifiable through electron microscopy and culturing of stool samples (Gillespie and Bamford, 2007).In the United States and infact, all over the world, few laboratories culture stool specimen routinely for E. coli O157: H7, resulting in the actual incidence of infection with this organism being unknown.

The number of outbreaks of E. coli O157:H7 infection reported to the Centre for Disease Control and Prevention (CDC) has increased to 30 in 1994 (Boyce et al., 1995) and may be more now. This increase may be attributed in part to increased reporting and screening of E. coli infection by laboratories.Malnutrition is the syndrome that resultsfrom the interaction between poor diets and diseases which leads to most of the anthropometricdeficits observed among children inless developed countries (WHO, 1995).

1.3 Aim
This study was aimed at determining the antibiogram of diarrhoeagenic Escherichia coliin diarrhoeic patients under-five years and their nutritional status.

1.4 Objectives 
The specific objectives of this work were to:

1. isolate and characterizeEscherichia coli O157, Verocytotoxigenic E. colifrom diarrhoeal stools of children under 5yrs in Kaduna State.

2. determine the antibiotic susceptibility pattern of the Escherichia coli isolates.

3. assess the nutritional status of the diarrhoeic children using Z-scores (internationally accepted reference standards).

4. obtain demographic data and risk factors associated with diarrhoea using pre-adopted questionnaire.

1.5 Research questions
1. Is the target organism present in the faeces of the children presenting with diarrhoea?

2. Is the organism isolated pathogenic?

3. Is the target organism susceptible or resistant to the antibiotics to be used in the study?

1.6 Research hypothesis
Ho:E. coli is not the causative agent of diarrhoea in children under five years.

H0: The organism isolated is not pathogenic in children under five years.

H0: The target organism is not susceptible or resistant to the antibiotics used.

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