Diarrhoea is defined as having loose or watery stools at least three times per day or more frequently than normal for an individual. More than 1 billion cases and at least 4 million deaths per year are attributed to diarrhoea worldwide [1]. In developing countries an estimated 1,000 million episodes occur each year in children below five years, causing 5 million deaths among them annually, out of which 80% occur in the first two years of life [2]. Fifteen countries contribute three quarters of childhood deaths due to diarrhoea in children under five years of age worldwide out of which India ranks first [3]. In India acute diarrhoeal diseases lead to 13% deaths in under five age group, during the year 2009, about 11.2 million cases with 1,762 deaths were reported [4]. According to a report by National Institute of Cholera and Enteric Disease, Kolkata, (India), crude death rate due to diarrhoea in rural India is 9.3 per 1,000 population and the diarrhoeal deaths account for 22% of total rural deaths among 0 to six years age group children [5]. According to annual Uttarakhand State Report 2009, out of 29 disease outbreaks, acute diarrhoeal disease and food poisoning contributed for 3 of them [6]. Rotavirus is the single most common pathogen which causes gastroenteritis in both developed and developing countries but the trend of bacterial enteropathogens vary largely between developed and developing countries [7]. Five types of Escherichia coli are responsible for as much as 25% of all diarrhoeal diseases in developing countries. Shigella species are responsible for 10 to 15% of acute diarrhoeas in children less than 5 years of age [8]. Other bacterial enteropathogens are Vibrio cholerae, Campylobacter jejuni, Aeromonas species, Bacteroides fragilis and Providencia alkalifaciens. The trends of bacterial enteropathogens causing gastroenteritis keep on changing with change in standard of living and environmental hygiene [7]. So the periodic renewal of the knowledge about trends of the bacterial enteropathogens is very essential. An epidemiologic study of an infectious disease in a community is always considered to be an initial step toward the introduction of the proper interventions for controlling the disease because the features and the patterns of isolation of etiologic agents of the disease vary from place to place depending on the local meteorology, geography, and socio-economic elements [9].
This study was conducted in Veer Chandra Singh Garhwali Government Medical Science & Research Institute, Srikot, Srinagar, Pauri Garhwal Uttarakhand, (India), which is a tertiary health care centre. This study aims at bridging the gap between the large number of diarrhoea patients and scanty data and knowledge about prevalent bacterial enteropathogens in Uttarakhand state.
Since diarrhoea is a disease in which the prevalence of different etiological agents vary largely on the basis of geographical region,socio-economic status, standard of living and environmental hygiene, henceforth the studies carried out in other places may not fit aptly in this hilly regions of Garhwal.
For earlier and the specific intervention for diarrhoea its very important to know the exact pathogen and its antimicrobial sensitivity in that particular area so that we can decrease the financial load on the patient as well as on health care. This study also takes an overview of the awareness-application gap for oral rehydration therapy among the population under study.
Methodology
This is a cross-sectional observational study including 280 individuals conducted at a tertiary health care centre from 1st June 2012 to 31st July 2012. The study was approved by Institutional Ethics Committee of the Institute patient with history of acute diarrhoea i.e. history of loose stools at least 3 times a day or increased frequency for the particular individual since less than 14 days of duration, patient should fall in the age group of 0-12 years of age group, He /she should have enrolled in out door patient department (OPD) or indoor patient department (IPD) of department of Pediatrics or Emergency department were enrolled for the study. The children with diarrhoea due to malnutrition and infective diarrhoea caused by other than bacteria were excluded from the study.
An informed consent was taken from parents or guardians after explaining them all prospects of study. A pre formed questionnaire was filled by asking some questions to the parents or guardians of the patient who included questions about the following:
Particulars of the patient.
A complete history of the duration, frequency, characteristics of diarrhoea and associated symptoms.
Preadmission treatment.
Awareness about oral rehydration therapy and whether patient started it or not.
Presence of any other case of diarrhoea in the family or locality of the patient.
Water source of the family and the method of drinking water storage.
A record sheet was filled by clinical examination of the patient which included.
General physical examination, vitals.
Assessment of nutrition.
Assessment of dehydration according to the description in Nelson’s Textbook of Pediatrics.
Sample Collection: Patient’s attendant was given a sterile wide mouthed container and completely explained to collect the stool sample.
Laboratory Analysis: The stool samples were sent to microbiology laboratory of the institute without using any transport media within 24 hours of presentation of the patient.
Stool sample was subjected to microscopy, culture and antimicrobial susceptibility test.
Rota virus-latex agglutination method.
Parasites-iodine wet mount.
For Escherichia coli sample was cultured on Bloodagar, On MacConkey’s medium, colonies are bright pink due to lactose fermentation. Growth is inhibited on selective media such as deoxycholatecitrate agar.
For Salmonellae spp-identified by no change in colour of Mac Conkey agar due to non lactose fermenting nature of its colonies. Enrichment media used was Selenite-F Broth.
For Shigella spp-colorless colonies on MacConkey agar. Deoxcholate citrate agar was used as a selective media
For Vibrio cholera - enrichment media used was Alkaline peptone water. Plating media used was TCBS medium
For identification of Klebsiella spp.- For identification of Klebsiella spp.- identified by its large mucoid colonies when grown on blood agar.
The patients with stool samples showing presence of virus or parasites were excluded from the study.
Further the samples were subjected for antimicrobial susceptibility with the help of Mueller Hinton agar using Kirby bauer-disk diffusion method .
Results
Epidemiological features:During two month study period, out of all the presented with acute diarrhoea, 280 children whose stool culture positive for bacteria were included in the study. The data collected by mean of structured study questionnaire revealed various epidemiological features of these children with acute diarrhoea and bacterial enteropathogens as etiological agent.
[Table/Fig-1] shows the pattern of age distribution among cases. Out of total studied cases maximum fall under the age group of 1-3 years i.e 100 (35.7%) followed by age group of 3-5 years (22.1%), least number of cases were seen in age group of 9-11 years (4.2%).
| Age group | No. of children (%) n=280 |
|---|
| <6 months | 26 (9.2) |
| 6 months- 1 year | 23 (8.2) |
| 1-3 years | 100 (35.7) |
| 3-5 years | 62 (22.1) |
| 5-7 years | 28 (10) |
| 7-9 years | 18 (6.4) |
| 9-11 years | 12 (4.2) |
About 54.6% of all the cases were males and 45.4% were females. Other epidemiological characteristics are shown by [Table/Fig-2].
Distribution of various epidemiological factors among the children presented with acute diarrhoea
| Epidemiological factor | No. (%) n=280 |
|---|
| Sex | Male | 153 (54.6) |
| Female | 127 (45.4) |
| Residence | Rural | 161 (57.5) |
| Urban | 119 (42.5) |
| Most frequently used water source | Gadhera (natural water spring) | 46 (16.4) |
| Water tank | 95 (33.3) |
| Old water tank | 56 (20) |
| Public hand pump | 44 (15.7) |
| Public tap | 39 (13.9 ) |
Parents of 263 (93.9%) were aware of Oral Rehydration Solution (ORS) therapy but was applied by parents of only 173 children (61.8%).
Prevalence of Bacterial Enteropathogens
Escherichia coliEscherichia coli was the most frequent enteropathogen isolated in diarrhoeal stool samples, being found in 124 samples (44.2%), 79 (28.2%) samples were positive for Shigella, 38 (13.6%) samples had shown Salmonella while Klebsiella and Campylobacter were positive in 22 (7.8%) and 17 (6.1%) samples. None of the sample was positive for Vibrio [Table/Fig-3].
Prevalence of various bacterial enteropathogens identified among children with acute diarrhoea
| Bacterial agent | No. (n=280) | %age |
|---|
| Escherichia coli | 124 | 44.2 |
| Shigella | 79 | 28.2 |
| Salmonella | 38 | 13.6 |
| Klebsiella | 22 | 7.8 |
| Campylobacter | 17 | 6.1 |
| Vibrio | 00 | 00 |
The age related distribution has shown that majority of the bacterial isolates have peak incidence between age 1-3 years being 34.7% for E.coli, 32.9% for Shigella, 50% for Salmonella, 52.9% for Campylobacter on the other hand, Klebsiella has peak incidence among infants (77.2%) [Table/Fig-4].
Age related prevalence of various bacterial enteropathogens causing childhood diarrhoea
| Age group | E. coli n=124 (%) | Shigella n=79 (%) | Salmonella n=38 (%) | Klebsiella n=22 (%) | Campylo-bacter n= 17 (%) |
|---|
| <6 months | 14 (11.4) | -- | -- | 12 (54.5) | -- |
| 6 months-1 year | 18 (14.5) | 7 (8.9) | 1 (2.6) | 5 (22.7) | 2 (11.8) |
| 1-3 years | 43 (34.7) | 26 (32.9) | 19 (50) | 3 (13.7) | 9 (52.9) |
| 3-5 years | 27 (21.7) | 18 (22.7) | 9 (23.7) | 2 (9.1) | 6 (35.3) |
| 5-7 years | 12 (9.6) | 14 (17.7) | 3 (7.9) | -- | -- |
| 7-9 years | 8 (6.5) | 8 (10.2) | 2 (5.3) | -- | -- |
| 9-11 years | 2 (1.6) | 6 (7.6) | 4 (10.5) | -- | -- |
E.coli is the only agent distributed among all age groups showing 25.8% incidence among infants, 56.5% between 3-5 years and least in age group >5 years (17.7%) [Table/Fig-4].
Both Shigella and Salmonella had least incidence among infants (8.9 % and 2.1% respectively) and their incidence increases with age showing peak between age group 1-5 years (55.7% and 73.6% respectively).
Both Klebsiella and Campylobacter had shown no isolates in age group >5 years.
Clinical Characteristics
[Table/Fig-5and6]
Antibiotic Resistance
[Table/Fig-7]
Discussion
Very few studies were conducted in India which focuses on pattern of bacteria as etiological agent in children presented with acute diarrhoea. This study covering a two month period was unique in its kind and was one of the very few studies which focused on epidemiological, microbiological and clinical aspects of childhood diarrhoea of bacterial etiology. The number of males suffering with diarrhoea were more in number than female with a ratio of 1:1.202 which is similar to the finding of the studies done over childhood diarrhoea [10–12].
Age distribution shows that children suffering from diarrhoea were 17.4% below 1 year of age, 57.85% were between 1 and 5 years. of age and 20.6% of more than 5 years of age [Table/Fig-1]. The maximum sufferers were between 1 and 3 years of age. A similar pattern of age distribution has been found in earlier done studies [13,11].
The study reveals that diarrhoea is more common in children living in rural set up (57.5%) than the one living in urban set up (42.5%) [Table/Fig-2]. This can be attributed to poor quality of hygienic condition and sanitation practices in rural population which contributes high risk of infection especially among children. Other epidemiological factors tend to play a role in increased susceptibility of infection to enteropathogen samong rural population. The main source of water available to rural people in present study area either natural springs or public handpumps, which are at more risk of contamination, resulting in outbreaks of diarrhoea.
Questionnaire also reveals that out of all 93.9% of the parents of patients under study were aware about the oral rehydration therapy while only 61.8% practiced it. An almost similar pattern has been found in a study [14,15]. The major reason behind this gap is considered lack of maternal knowledge towards diarrhoea and oral rehydration therapy [14].
In our study Escherichia coli was recorded as the predominant bacteria with 44.2% of prevalence followed by Shigella, Salmonella, Klebsiella and Campylobacter with 28.2%, 13.6%, 7.8% and 6.1% respectively [Table/Fig-3]. This pattern was similar to other studies done in India by workers like Joshi et al., and Subhash kumar et al., [16,17] which had shown higher incidence of diarrhoeagenic E.coli, supporting the well-documented role of E.coli spp. in diarrhoeal disease [18]. However, this pattern is in contrast to other areas of Africa and Malaysia [10], which had reported E.coli as least common etiological agent causing agent, which can be because of alteration in epidemiological features of host as well environment.
Of all the bacterial diarrhoeal cases 36.42% were from age group 1-3 years. This can be attributed to negligent attitude of parent’s toward children of this age group and increased mobility of children due to their recently acquired walking ability. Escherichia coli has been found to be one of the causative agents at all age groups. Klebsiella mostly affected children of age group less than six months. Shigella and Salmonella are least likely to cause infantile diarrhoea but their incidence increases with age. These findings are consistent with those of African studies [19,20]. This might be so because Salmonella are least likely to adapt to domesticated cattles and all varieties of fowls and chickens. The reservoir of infection in animals constitute the principal source of diarrhoea caused by Salmonella spp [21]. Camplylobacter and Klebsiella were the causative agent till the age of five years only [Table/Fig-4]. The higher incidence of Campylobacter among children do not match with findings shown by Gascón et al,. and Lindblom et al., [22,23] who described low incidence among children but our findings matches to greater extent with work done by NguendoYongsi who had shown higher prevalence of Campylobacter among children <5 years [11].
Fever was found to be a predominant clinical feature present in 89.6% of patients of diarrhoea. All 100% patients who had diarrhoea due to Salmonella suffered from fever, followed by patients of diarrhoea due to Escherichia coli (93.5%), Shigella (91.1%), Klebsiella (72.7%) and Campylobacter (52.9%) [Table/Fig-5]. From the above finding it can be deciphered that fever is a common clinical feature in diarrhoea with Salmonella occupying the majority with all the cases presenting with fever.
Clinical characteristics of children with acute diarrhoea and their presentation depending upon the various bacterial etiological agent
| Clinical characteristic | n=280 (%) | E. coli n=124 (%) | Shigella n=79 (%) | Salmonella n=38 (%) | Klebsiella n=22 (%) | Campylo-bacter n= 17 (%) |
|---|
| Fever | 251 (89.6) | 116 (93.5) | 72 (91.1) | 38 (100) | 16 (72.7) | 9 (52.9) |
| Vomiting | 173 (61.8) | 55 (44.4) | 61 (77.2) | 32 (84.2) | 13 (59.1) | 12 (70.6) |
| Frequency of diarrhoea | <5 times /day | 147 (52.5) | 102 (82.3) | 8 (10.1) | 2 (5.3) | 19 (86.3) | 16 (94.1) |
| 5-10 times/day | 117 (38.2) | 22 (17.7) | 73 (92.4) | 15 (39.5) | 3 (13.6) | 1 (5.9) |
| >10 times /day | 23 (8.2) | -- | 2 (2.5) | 21 (55.2) | -- | -- |
| Degree of dehydration | No dehydration | 65 (23.5) | 44 (35.5) | 12 (15.2) | -- | 5 (22.7) | 4 (23.5) |
| Some dehydration | 188 (67.1) | 80 (64.5) | 60 (75.9) | 18 (47.4) | 17 (77.3) | 13 (76.5) |
| Severe dehydration | 27 (9.6) | -- | 7 (8.7) | 20 (52.6) | -- | -- |
Vomiting was found less common i.e., only in 61.8% of the diarrhoea patients. Again 84.2% of the patients with diarrhoea due to Salmonella showed vomiting as a clinical feature while in Shigella (77.2%), Campylobacter (70.6%), Klebsiella (59.1%) and Escherichia coli (44.4%) lesser number of patients presented with vomiting. This above mentioned finding again denotes the more severe nature of diarrhoea caused due to Salmonella [Table/Fig-5].
Maximum number of the patients (52.5%) presented with frequency of diarrhoea less than 5 times a day. Least number of the patients (8.2%) presented with frequency of more than 10 times a day. And rest 38.2% lied in the frequency of in between 5 and 10 times a day. Among the patients who presented with the frequency of diarrhoea less than 5 times a day, maximum number (86.3%) was contributed by Klebsiella. Shigella contributed maximum number (92.4%) to those who presented with frequency of 5-10 times per day. And Salmonella was the major (55.2%) agent to those who presented with frequency of stools more than 10 times a day [Table/Fig-5].
Out of all 67.1% children presented with some dehydration followed by no dehydration (23.5%) and severe dehydration (9.6%). Out of 124 patients of Escherichia coli 80 patients presented with some dehydration and rest with no dehydration. While the patients with Salmonella as etiology suffered more with severe dehydration (52.6%) [Table/Fig-5].
Out of 280 patients maximum number of patients came after 3-5 days of illness. Escherichia coli, Shigella and Salmonella induced diarrhoea were mainly made the patient to come to the hospital after 3-5 days while in case of Klebsiella the maximum patients came after 5-7 days of illness which may be explained by the mildness of symptoms caused due to Klebsiella [Table/Fig-6].
Duration of Ilness among cases and its relation to various etiological agents causing childhood diarrhoea:
| Duration | n=280 (%) | E.coli n=124 (%) | Shigella n=79 (%) | Salmonella n=38 (%) | Klebsiella n=22 (%) | Campylobacter n=17 (%) |
|---|
| ≤ 1 day | 27 (9.6) | 9 (7.2) | 4 (5.1) | 5 (13.2) | 4 (18.2) | 5 (29.4) |
| 1-3 days | 35 (12.5) | 10 (8.1) | 13 (16.4) | 7 (18.4) | 3 (13.6) | 2 (11.8) |
| 3-5 days | 114 (40.7) | 52 (41.9) | 37 (46.8) | 17 (44.7) | 4 (18.2) | 4 (23.5) |
| 5-7 days | 64 (22.8) | 25 (20.2) | 19 (24.1) | 7 (18.4) | 10 (45.5) | 3 (17.6) |
| >7 days | 40 (14.2) | 28 (22.5) | 6 (7.6) | 2 (5.3) | 1 (4.5) | 3 (17.6) |
[Table/Fig-7] shows the antimicrobial resistance pattern among all the pathogens that were isolated. The pattern of antibiotic susceptibility of various bacterial pathogens matches to greater extent to work done by Daniel R., and Diniz-Santos, et al., [24]. High resistance to Co-trimoxazole among all pathogens can be attributed to its injudicious use among the population because of its wide antimicrobial spectrum most likely to be used for various infections.
Antimicrobial resistance of bacterial specimens isolated from diarrhoeal stool
| Antibiotic | E.coli n=124 (%) | Shigella n=79 (%) | Salmonella n=38 (%) | Klebsiella n= 22 (%) | Campylobacter n= 17 (%) | n= 280(%) |
|---|
| Amoxicillin + clavulanic acid | 12 (9.6) | 14 (17.7) | 2 (5.2) | 1 (4.5) | 1 (5.8) | 30 (10.7) |
| Ampicillin+ sulbactam | 22 (17.7) | 10 (12.6) | 3 (7.8) | 2 (9.1) | 2 (11.8) | 45 (16.1) |
| Cefuroxime | -- | -- | -- | -- | -- | -- |
| Co-trimoxazole | 37 (29.8) | 54 (68.4) | 9 (23.6) | 14 (63.6) | 9 (52.9) | 123 (43.9) |
| Cefotaxime | -- | -- | -- | -- | -- | -- |
| Aztreonam | -- | -- | -- | -- | -- | -- |
| Piperacillin-tazobactam | 8 (6.4) | 5 (6.3) | 7 (18.4) | 8 (36.4) | 1 (5.8) | 24 (8.5) |
| Ciprofloxacin | 4 (3.2) | 9 (11.4) | 10 (26.3) | 7 (31.8) | 3 (17.6) | 25 (8.9) |
Considerable resistance rate has been found to Co-trimoxazole, Amoxiclav, ampicillin associated with sulbactam among Shigella specimens. Separate assessment of antimicrobial resistance patterns indicates that Shigella is more difficult to treat and requires careful consideration at the choice of antimicrobial therapy. Thus, use of these drugs is not recommended for the empirical treatment of acute bloody diarrhoea in children.
In very severe cases, with evidence of dissemination of disease, intravenous third generation Cephalosporins like cefotaxime and cefuroxime is the best choice for many, while other drugs can be adequate choices for the empirical treatment of severe acute diarrhoea in children.
Salmonella strains were found to be multi-susceptible, as high rates of resistance were not found against any of the drugs, except some resistance 26.3% was found to be against ciprofloxacin.
This resistance to ciprofloxacin among non-typhoidal Salmonella strains isolated from stool specimens has already been reported from several parts of the world [25,26].
Oplustil et al., did not isolate any strain of Salmonella resistant to fluoroquinolones from blood samples [27]. However, Salmonella spp. strains isolated from blood samples are recognized to be more susceptible than those from stools [28].
E. coli isolates were found to have a high rate of resistance to ampicillin, ampicillin + sulbactam and Co-trimoxazole, this can be explained as E.coli being so common agent of diarrhoea, its strains are most mostly exposed to different antibiotics injudiciously.
The knowledge of resistance pattern of common etiological agents in local area can help practitioners to choose an adequate antimicrobial drug to start empirical therapy in a patient with severe diarrhoea without knowledge of a specific pathogen. However, as soon as the results of stool cultures are available, the therapy can be altered to a safer drug based on the antimicrobial susceptibility pattern.
Conclusion
As per the previous records Rotavirus caused majority of the childhood diarrhoea case which led to overlooking of bacterial diarrhoeal cases. This study was aimed at finding the prevalence of bacterial diarrhoea among childhood patients. Escherichia coli was found most common bacteria among total cases followed by Shigella, Salmonella, Klebsiella and Campylobacter. It was interesting to note that not only one but different types of bacteria were isolated from the stool sample. More studies should be carried out to prevent the disease in its primordial stage. Comparative studies related to causative agents like bacteria, viruses should be given more importance because they help in disease prevention strategies. Role of Information Education and Communication is very important regarding diarrhoeal and hence should be prioritized.