Microbial aetiology of diarrhea in children less than five years of age at tertiary care hospital of Western Uttar Pradesh, India

Introduction and Aim: Diarrhoeal diseases are one of the major causes of childhood morbidity and mortality globally. Multiple aetiologies and associated risk factors are responsible for diarrhoeal diseases in children. The present study aims to determine the microbial aetiology and possible risk factors of diarrhoea in children less than five years of age at Chhatrapati Shivaji Subharti Hospital, Subharti Medical College, Meerut in Western Uttar Pradesh, India. Materials and Methods: A total of 240 children < 5 years of age with history of acute diarrhoea of less than 7 days duration were included in the study. Microbial etiology of diarrhoea in children were looked for. Bacterial etiology was identified by standard bacteriological technique. Diarrheagenic E. coli (DEC) were identified by gene-specific polymerase chain reaction. Rotavirus (RV) antigen was detected by Enzyme Immunoassay (EIA), parasites were identified by wet mount preparation and fungal etiology was identified by microscopy and culture on Sabouraud’s Dextrose Agar. Results: Out of 240 stool samples, 152 were identified as known aetiology of diarrhea. Microbial etiology could be established in 133/240 (55.41%) stool samples. The most common microbial etiology was bacterial 63(47.36%) followed by rotavirus 35 (17.29 %), parasitic etiology in 22 (16.54%) and least common etiology was fungal in 13 (9.77%) cases. Among other etiology, Lactose intolerance was found in 19/240 (7.91%) cases. Mixed aetiology with two or more pathogen causing diarrhoea was seen in 27% cases. Conclusion: Aetiological agents of diarrhoea could be identified in 63.3% cases. Causes of childhood diarrhoea is polymicrobial and preventable. Awareness and education especially to the mothers regarding the disease, safe drinking water, proper food intake and proper sanitation are key to overcome the burden of the preventable infectious diseases among children less than five years of age.


INTRODUCTION
iarrhoea is the leading cause of childhood morbidity and mortality worldwide and is responsible for killing around 525 000 children every year (1).Majority of childhood death due to diarrhoea occur because of inadequate availability of clean drinking water and lack of access to sanitation (1,2).Diarrheal diseases are mainly caused by microorganisms which includes bacteria such as diarrheagenic E. coli (DEC) , Campylobacter jejuni, Salmonella, Shigella and Vibrio, parasites such as Giardia lamblia , helminths, cryptosporidium species, and nematodes, viruses like rotavirus (RV), adenovirus, hepatitis A, E and Norwalk viruses.DEC and RV are the commonest cause of diarrheal infection in children and responsible for most of the outbreaks.(3,4).Though all these organisms are transmitted through contaminated food and water they differ in the exact infectious dose needed to cause the illness (5).Infectious diarrhea occurs as a result of two major disturbances in normal intestinal physiology, i.e., increased intestinal secretion of fluid and electrolytes, predominantly in the small intestine and decreased absorption of fluid, electrolytes and sometimes nutrients involving the small and large intestine.Infectious diarrhea typically shows up as one of three major clinical types: i) acute watery diarrhea (which usually resolves within 5-10 days), ii) diarrhea with blood (dysentery) and iii) persistent diarrhea (lasting for more than 14 days).The etiology of childhood diarrhoea varies from region to region and studies focussing on multiple aetiologies of diarrhoea in children are limited.(6,7,8).Paucity of data from this geographical area prompted us to carry out this study D to determine the different aetiologies of diarrhea in children below the age of five years.

MATERIALS AND METHODS
This cross-sectional study was conducted in Chhatrapati Shivaji Subharti Hospital a tertiary care teaching Hospital, Meerut, UP, India from November 2017 to December 2019.A total of 240 stool samples were collected from children <5 years of age with history of acute diarrhoea of less than 7 days duration and fulfilling the diarrheal case definition as per World Health Organization (WHO) guidelines was included (2) .Children with history of diarrhoea associated with blood, children who were immunocompromised or on immune-suppressive therapy and prolong steroid treatment were excluded.Informed consent was obtained from parents/guardians before collection of clinical samples.Approval from the Institutional Ethics and Research Committee was obtained before the commencement of the study.

Identification of bacterial aetiology
The stool samples were directly inoculated onto MacConkey agar, deoxycholate citrate agar (DCA), Columbia blood agar media and Thiosulphate-citrate bile salts-sucrose agar (TCBS) and incubated overnight at 37 0 C and processed as per standard bacteriological technique (8,9).The colonies isolated were identified by colony characteristics, standard biochemical tests (Indole/Methyl red/Voges-Proskauer/Citrate (IMViC), Urease, Oxidase and Triple sugar Iron test), motility test and agglutination with specific antisera (9,10).Molecular confirmation of Enterotoxigenic E. coli (ETEC), Enteropathogenic E. coli (EPEC), Enterohemorrhagic E. coli (EHEC) and Enteroinvasive E. coli (EIEC) strains were identified using PCR primers that targeted virulence genes (elt, estA, eae, bfp, stx-1, stx-2 and ipaH) and EAEC strains were identified based on the presence of the aggregative adherence plasmid region (pAA).DNA or RNA was extracted from culture positive DEC strains by using Qiagen mini-DNA/RNA extraction kit USA and the first strand cDNAs were synthesized from the extract and the cDNA and DNA were both stored at -20 0 C. The multiplex PCR was performed to detect the of ETEC, EPEC, EHEC and EIEC strains of E. coli (7,9,10).

Identification of parasitic aetiology
The stool samples were examined macroscopically to look for parasites and segments following which microscopic examination was carried out by direct wet mount (saline and iodine) for presence of trophozoites, cyst and ova using low power (10X) and high-power (40X) objectives (10,11).

Identification of fungal aetiology
Fungi were identified on the basis on direct microscopy by Gram stain and colony characteristics on Sabouraud's Dextrose Agar.Candida species isolated was further identified by Germ tube test (9,11).

Identification of viral aetiology
All the samples were subjected for rotavirus antigen detection using commercial Enzyme Immuno-Assay (EIA) kit (Premier Rotaclone meridian Bioscience Catalog: 696004, Cincinnati, USA).This test utilizes monoclonal antibodies against VP6 antigen (group specific antigen for all known human rotaviruses;12).

Lactose intolerance tests
Lactose intolerance was diagnosed by Lactose tolerance test and Benedict's test (3).

Statistical analysis
The findings were analyzed using master excel sheet (MS office 2010) and statistical analysis was done accordingly.
Fig. 2: Types of diarrheagenic E. coli detected by PCR Distribution of various types of DEC is shown in Figure 2. EPEC was the commonest (51%) followed by ETEC (27%), EAEC (14%) and EHEC (8%).Mixed aetiology with two or more pathogen causing diarrhoea was seen in 27% cases.Among the RV positive cases 6 cases had co-infections with DEC, 4 with Candida albicans, 3 with Klebsiella spp.and one with Campylobacter spp.Similarly, DEC along with RV had co-infection with Giardia lamblia in 3 cases and C. albicans in 2 cases.
Majority of cases of diarrhoeal diseases was seen in the age group of 13-24 months (33.5%) followed by 7 -12 months (26.3%).Lowest numbers of cases were reported in 0-6 month's age group (16.4%).Age wise distribution of various etiological agents is shown in Table 3. Lactose intolerance was the most common cause of diarrhoea in 0-6 months age group 14 (9.2%) followed by bacterial infection 8 (5.2%).In most of the other age group bacterial aetiology is the commonest followed by viral.The various risk factors associated with cases of diarrhoea is elaborated in Table 4.There was male predominance 86 (56.5%) and the male to female ratio was 1.3:1.A total of 47 (30.9%) children were malnourished and malnutrition was maximum in children suffering from bacterial infection and minimum in children with parasitic infection.The diarrhoeal disease was more common in rural population (57.8%) and in the low-income group (LIG) (44%).However, the infection was least in high-income group (HIG) (21%).
Fever (60.5%) was the most common clinical presentations seen in cases of diarrhoea followed by dehydration (42.7%) and vomiting in (29.6%).Abdominal pain was the least common clinical symptoms reported in (7.2%) cases.The etiology wise clinical presentation of children suffering from diarrhoea is shown in Table 5.

DISCUSSION
Childhood diarrhea is one of the common causes of morbidity and mortality worldwide.
Mixed aetiology with two or more pathogen causing diarrhoea was seen in 27% cases.Even among the mixed aetiology bacterial infections was the commonest followed by RV.These mixed infections are associated with prolonged disease and more severe clinical presentation.The role of mixed infection also depends on microbial interaction carriage and invasion of pathogens which is directly related to host response pattern of single vs. multiple infections, required to elicit synergy or inhibition.Mixed infection of etiological agents might be complementing each other's strategies of pathogenesis resulting in an increased disease severity.Shrivastava et al. reported mixed infection in 30% cases of diarrhea (6).
Various risk factors were identified in our study.The rate of the diarrhoea was much higher in male children (56.5%)This finding is in good agreement with previous studies (4,5,13).The positivity rate was high in 13-24 months age (< 2 years) and lowest in < 6 months of age group.In both the age group, bacterial infections was the leading cause seen in 45% & 42.5% cases followed by rotavirus in 29% & 27.5% cases respectively.Children between 7 months to 2 years was the most susceptible age group similar findings were also reported by other studies.(3,14,15).The diarrhoeal disease was more common in rural population (57.8%) which indicates that that rural population was more vulnerable to infection.Similar findings have been reported in the past (6,16).We observed a significant correlation between diarrhoea and malnutrition in 31% children.Malnutrition is a major risk factor that contributes to childhood diarrhea.Similar correlation between diarrhoea and malnutrition was shown by Nimri et al.,in 25% children (16).Socio-economic status is an important risk factor of diarrheal infections and majority of children in our study group belonged to LIG (44%).Similar finding was reported by Wang et al., from Beijing.They showed that children from poorer provinces living with poor health and environmental conditions were more prone to diarrheal infection (10).
Thus providing basic facilities such as clean water supply and sanitation is crucial to enhance the hygiene level of communities (4).
Physical examination is vitally important in assessing the individual's hydration status and in identifying other causes and risk factors for diarrhoea.In this study standard clinical criteria have been established to formally assess the severity of diarrhea on the basis of frequency of episodes of diarrhoea, fever, abdominal pain and hydration status in infants and young children and it proved to be helpful to guide the appropriate diagnosis and treatment.Vomiting and dehydration were the commonest clinical presentations predominantly seen in cases of rotavirus associated diarrhoea.Similarly a study by Saeed et al., showed vomiting and dehydration in 55% and 60% of RV positive cases respectively (5).Studies have shown that RV associated gastroenteritis results in dehydration with shock, electrolyte imbalance and increases body temperature up to 102 0 F, with eventual hospitalization (3).It has been seen that water-borne and faeco-oral route of infections, unhygienic practices, poor sanitations and lack of awareness regarding preventable diarrheal diseases are the major risk factors involved in diarrhoea (4,15,17).The identification of etiological agents of diarrhoea in children and the associated risk factors could help in prevention and management of diarrhea in children.

CONCLUSION
The infectious aetiology of diarrhoea in children < 5 years of age is polymicrobial.Identification of possible causes of diarrhoea and various risk factors could help in management of diarrhoeal diseases in children.Contaminated food and water are the main vehicle of transmission therefore awareness and education especially to the mothers regarding the disease, safe drinking water, proper food intake and proper sanitation are key to overcome the burden of this infection.Large scale multifactorial epidemiological studies on diarrhoeal diseases in children should be carried out to know the exact burden in future.

Fig. 1 :
Fig. 1: Various aetiology of diarrhea in children less than 5 years of age (n=152)

Table 1 :
Microbial aetiology of diarrhoea in children <5 years of age (n=133)

Table 2 :
Distribution of various bacterial and parasitic agents identified in cases of diarrhoea

Table 3 :
Age-wise distribution of various aetiological agents in children with diarrhoea

Table 4 :
Various risk factors associated with cases of diarrhoea (n=152)

Table 5 :
Aetiology wise clinical presentation of children suffering from diarrhoea