|Year : 2016 | Volume
| Issue : 3 | Page : 375-379
A cross-sectional study on aetiology of diarrhoeal disease, India
S Purwar1, D Bhattacharya2, SC Metgud3, D Kumar4, SD Chitambar5, S Roy4
1 Department of Microbiology, All India Institute of Medical Sciences, Bhopal, Madhya Pradesh; Department of Microbiology, KLE University's, J N Medical College, Belagavi, Karnataka, India
2 Regional Medical Research Centre (ICMR), Belagavi, Karnataka; Regional Medical Research Centre (ICMR), Bhubaneswar, Odisha, India
3 Department of Microbiology, KLE University's, J N Medical College, Belagavi, Karnataka, India
4 Regional Medical Research Centre (ICMR), Belagavi, Karnataka, India
5 National Institute of Virology (ICMR), Pune, Maharashtra, India
|Date of Submission||30-Oct-2014|
|Date of Acceptance||06-Jul-2015|
|Date of Web Publication||12-Aug-2016|
Regional Medical Research Centre (ICMR), Belagavi, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Global, regional and national estimates clearly place diarrhoeal diseases as a major, albeit to an extant neglected public health problem. Deaths of children aged <5 years owing to diarrhoea was estimated to be 1.87 million at the global level (uncertainty range from 1.56 to 2.19 million), which is approximately 19% of total child deaths. Objectives: The present report is a cross-sectional study undertaken to estimate the role of various aetiological agents causing diarrhoea in North Karnataka and adjoining areas of Maharashtra and Goa. Methods: Three hundred stool samples were collected from patients seeking health care at KLES Dr. Prabhakar Kore Hospital and Medical Research Centre, Belgaum; and processed for detection of various bacterial, viral and parasitic agents. Results: Bacterial pathogens attributed to 65.7% of diarrhoea cases, followed by viral infection (22%), parasitic infection (16.3%) and infection by Candida spp. (5.6%). The study identified Escherichia coli in general and Enteropathogenic E. coli in particular, and Group A Rotavirus to be the most frequently isolated pathogens among diarrhoea patients. Conclusion: The data generated from the current study will help the health officials for better interventional and treatment strategies for diarrhoeal diseases.
Keywords: Bacteria, diarrhoea, diarrhoeagenic Escherichia coli, Group A Rotavirus, ELISA
|How to cite this article:|
Purwar S, Bhattacharya D, Metgud S C, Kumar D, Chitambar S D, Roy S. A cross-sectional study on aetiology of diarrhoeal disease, India. Indian J Med Microbiol 2016;34:375-9
|How to cite this URL:|
Purwar S, Bhattacharya D, Metgud S C, Kumar D, Chitambar S D, Roy S. A cross-sectional study on aetiology of diarrhoeal disease, India. Indian J Med Microbiol [serial online] 2016 [cited 2019 May 21];34:375-9. Available from: http://www.ijmm.org/text.asp?2016/34/3/375/188358
Infections leading to diarrhoea continue to be major causes of childhood mortality, as well as morbidity in older age groups in developing countries. The proportion of deaths attributed to diarrhoea among children of 1-59 months of age is estimated to be 25% in Africa and 31% in South Asia.  Knowledge of the aetiology of acute diarrhoea is relevant for planning diarrhoeal disease control strategies, especially vaccine development.  There is no well-established vaccine against infectious diarrhoeal pathogens which is in significant developmental stage, barring Rotavirus vaccine which is much in use in many of the countries of western hemisphere and a strong candidate for its inclusion in government sponsored universal immunisation programmes. In comparable aetiological surveys carried out in developing countries, the rate of positive identification of microorganisms has been slightly lower, and when compared with viruses the role of bacterial agents has been greater ,,, although this could also be attributed to difficulty in virus identification and lack of virus detection/isolation facilities in resource poor settings. Moreover, in developing countries it has been recognised that enteric pathogens can frequently be encountered also in healthy children, making it more difficult to determine their true aetiological role in causing diarrhoea.  Furthermore, in developing countries it is not uncommon to isolate more than one enteric pathogen from the same child. , Mortality due to diarrhoea has declined over the past two decades from an estimated 5 million deaths among children under-five to 1.5 million deaths in 2004,  which parallels downwards trends in overall under-five mortality during this period.  Despite these declines, diarrhoea remains the second most common cause of death among children under-five globally.  India features at the top of the list of developing countries with an estimate of 3.86 lakhs annual child deaths due to diarrhoea.  In the South-East Asian region, almost 48% of the estimated 3.07 million deaths annually are attributed to acute respiratory infections and diarrhoeal diseases with the highest burden of diarrhoeal disease in five countries: Bangladesh, India, Indonesia, Myanmar, and Nepal where these diseases caused 60,000 deaths annually. , In this backdrop the present study was designed and conducted at Belgaum to understand the aetiology of diarrhoeal disease in North Karnataka and adjoining areas of states of Maharashtra and Goa. There are very few reports from Goa about Enteroaggregative E. coli (EAEC) which is an emerging pathogen. For other diarrhoeagenic aetiological agents comprehensive data from this area is not available in scientific publications. The infective aetiology of diarrhoea is usually not determined and decision of prescribing antibiotics by attending physicians is more often than not arbitrary without microbiological evidence. The viral diarrhoea will not require antibiotics but diarrhoea due to parasitic causes and invasive E. coli, Shigella will need antiparasitic/antibiotics, respectively. The data arising from our study will assist health care providers in being appraised of proportion of diarrhoea with infective aetiology and proportion of parasitic/viral/bacterial diarrhoea in this region. This will also help physicians in making an informed choice for prescribing antiparasitic/antibiotic or managing diarrhoea cases with rehydration therapy only.
Most of the studies either target one age group (like paediatric population) or one of the pathogens class (viral/bacterial/parasitic) or even one of the pathogens like Rotavirus or one of the diarrhoeagenic E. coli (DEC) like enteropathogenic E. coli (EPEC) in children/enterotoxigenic E. coli (ETEC) in travellers. In our study, diarrhoea patients of all ages were included and every stool sample was screened for parasitic, bacterial and viral causes. Then each E. coli isolate was identified to diarrhoeagenic pathotypes namely EPEC, ETEC, enteroinvasive E. coli (EIEC), Shiga toxin producing E. coli (STEC) and EAEC by polymerase chain reaction (PCR). The data thus generated provide comprehensive information about major causes of viral/bacterial and parasitic causes in the region.
A total of 300 stool samples were collected and processed from diarrhoea patients attending the IPD and OPD of KLES Dr. Prabhakar Kore Hospital and Regional Medical Research Center (ICMR), Belgaum, following standard protocol.  However, the majority of patients included in the study belong to the admitted cases. The study was approved by Institutional Ethics Committee of J. N. Medical College, KLE University. Subsequent to collection of diarrhoeic stool sample in a transparent, sterile, screw-capped 20 ml capacity vial, one small portion of faecal sample was separated for stool microscopy (including wet mount and iodine mount) to detect trophozoite, ova, cyst and for presence of large number of budding yeast cells. Another small portion was inoculated on Mac Conkey agar, Hektoen enteric agar, Thiosulphate citrate bile salt sucrose agar and Blood agar plates (Hi-Media, Mumbai) for culture and identification of bacteria.  The suspected bacterial colony after overnight incubation at 37°C was subjected to biochemical characterisation and identification and serotyping using group-specific antisera (Denka Seiken Co., Ltd., Tokyo., Japan). The E. coli isolated on culture plate from a diarrhoea stool cannot be interpreted as diarrhoeagenic without being identified as one of the diarrhoeagenic pathotypes. In most of the laboratories the E. coli is not subjected to further identification and at times considered pathogenic or part of commensal flora depending upon age of the patient. Occasionally the isolate is subjected to serotyping with one/two antisera which is grossly insufficient. The serotyping also at times results in overlapping hence does not provide accurate identification. We have targeted specific virulence genes for achieving differentiation amongst E. coli isolates. Our study provide exact incidence of EIEC and STEC which are quite often identified by conventional methods as Shigella or nonlactose fermenting E. coli which is incorrect. With this study, we emphasize to identify the E. coli from diarrhoea stool samples to various pathotypes by PCR and not consider as normal flora unless proved otherwise.
Rapid lysate of the culture concentrate grown in peptone water was used as template in PCR for detection of various DEC.  Subsequent to removing the part of stool sample for aforementioned tests the vials containing the stool samples were stored at −20°C. Samples stored at −20°C were subsequently subjected to detection of Group A Rotavirus (GARV) by ELISA and RT-PCR for Group B Rotavirus (GBRV), Enterovirus and Norovirus,, at National Institute of Virology, Pune.
E. coli and GARV were the major diarrhoeal pathogens detected in the age group of 0-4 years. No parasitic or Candida infection was observed in this age group. Apart from E. coli, viral enteric pathogens accounted for majority of diarrhoeal cases in the age group of 0-4 years. One hundred and ninety-seven bacterial isolates were obtained from 300 diarrhoeal patients giving a proportional aetiology of 65.7% bacterial diarrhoea in this region [Table 1]. Among the bacterial pathogens, pathogenic E. coli accounted for majority of cases (161, 53.7%) followed by Salmonella spp. (19, 6.3%), Shigella spp. (11, 3.6%) and Vibrio cholerae (6, 2%). Parasitic and Candida infection were detected among 49 (16.3%) and 17 (5.6%) of the diarrhoeal cases, respectively [Table 1]. Among parasitic infections, protozoa cysts/trophozoites and ova of helminths were detected in 2.6% and 13.6% of diarrhoeal pathogens, respectively. Viral infection was observed in 66 (22%) patients. Mixed or co-infection was observed in 50 (16.7%) cases [Table 2].
Among the E. coli isolates, EPEC accounted for majority of cases (67, 41.6%), followed by STEC (46, 28.6%), ETEC (27, 16.8%), EIEC (11, 6.8%) and EAEC (1, 0.6%) [Table 1]. Co-infection by STEC and ETEC was observed in 7 (4.3%) cases, and STEC and EAEC were observed in 2 (1.2%) cases. Among the enteric viruses, GARV accounted for majority (34, 11.3%) of diarrhoea cases, followed by Enterovirus (19, 6.3%), GBRV (8, 2.7%) and Norovirus (5, 1.7%) [Table 1]. Co-infection with pathogenic E. coli and viruses was detected among 39 (24.2%) cases [Table 2]. GARV was found to be a major pathogen (16, 41%) associated with co-infection, followed by Enteroviruses (8, 20.5%). Two individuals were found to be infected by both GARV and GBRV. Contrary to assumptions and available literature, GBRV was also seen as a sole diarrhoeagenic pathogen in children <4 years of age.
Our study also provides information about the co-infections by more than one virus and virus + bacterial co-infections also. None of the samples positive for parasitic or bacterial infection except E. coli showed co-infection with other viruses. Thirteen samples were detected with co-infection by two viruses at the same time [Table 2]. Mixed or co-infection was observed among 50 (16.7%) cases. As observed in other studies also  GARV infection was higher in the age group of 0-4 years.
Rotavirus and E. coli were the most common enteric pathogens causing diarrhoea that required hospitalisation with the former dominating during winter months and the latter in the other months. In the present study, bacterial pathogens attributed to almost 65% of diarrhoea cases, followed by viral infection (22%), parasitic infection (16.3%) and infection by Candida spp. (5.6%). The study identified E. coli in general and EPEC in particular and GARV to be the most frequently isolated pathogens among patients hospitalised for diarrhoea. Earlier studies reported that among hospitalised diarrhoeal children up to 5 years of age, DEC detection was high next to Rotavirus and in patients more than 5 years of age; occurrence of DEC-mediated diarrhoeal infection was next to cholera.  However, in the current study, DEC accounted for majority of the diarrhoea cases irrespective of age group. The prevalence of DEC in this study was almost 53.7%, which is more than the other reports from developing countries. ,,,
EPEC which is held responsible for diarrhoea in children was identified (rather the most common pathotype) in older population also (>10 years of age) which is contrary to current knowledge. Similarly, none of the patients was travellers per se but ETEC was found in 16.8% of diarrhoea cases. This is also not in agreement with current perception. It suggests entrenchment of ETEC in general population and that immunity to ETEC is not as strong as to confer immunity to resident population since ETEC is isolated from all age groups.
Diarrhoea incidence remains a tremendous burden on children in low- and middle-income countries, and diarrhoea has been shown to have a lasting influence on nutritional status in that an increase in diarrhoea prevalence has been linked to an increased risk of stunting. 
Viral and bacterial intestinal infections are common during early childhood and have been the object of numerous studies, but dual infections are not expected and are often misdiagnosed.  Dual infections raise the question of the possible role of each individual pathogen in causing the illness, or whether several pathogens act in synergy. However studies must be undertaken to obtain a better understanding of these infections.
The present study suggests that DEC is one of the important pathogens that should be considered in the surveillance programme of diarrhoeal diseases. Though most diarrhoea episodes are self-limiting and dehydration can usually be controlled with oral rehydration therapy, it would be ideal to be able to prevent diarrhoea, especially the more severe episodes which have a higher likelihood of progressing to complications or death. The data generated from the current study will help the health officials in betterment of treatment strategies for diarrhoeal diseases. Some prevention strategies such as improved water and sanitation and basic hygiene practices are generalisable and thus do not require knowledge of diarrhoea aetiology, but others such as vaccine programmes would benefit immensely from a comprehensive understanding of the overall burden of pathogen-specific diarrhoeal disease. The extent of water-borne infections shall invoke and assist health planners of relatively progressive states to take corrective steps in ensuring uncontaminated water supply.
The study also underlines the fact that in states such as Karnataka, Goa and Maharashtra also which have better health indicators, diarrhoea with infective aetiology is a problem which needs to be addressed.
Limitations of the study
Coccidian parasites were not looked in stool samples because they are usually more commonly seen in patients with chronic diarrhoea/immunocompromised patients. Theoretically very small probability of co-infection with one of the DEC and Salmonella/Shigella/Vibrio/parasitic/fungal pathogens exists. However, it was not determined for the very same reason of extremely low probability. Co-infections of viruses + parasites/E. coli + parasites and E. coli + other bacterial enteric pathogens were not looked into.
The authors are thankful to KLE University and Indian Council of Medical Research.
Financial support and sponsorship
Internal funds of KLES Dr. Prabhakar Kore Hospital and Medical Research Centre and Indian Council of Medical Research.
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Walker CL, Aryee MJ, Boschi-Pinto C, Black RE. Estimating diarrhea mortality among young children in low and middle income countries. PLoS One 2012;7:e29151.
Boschi-Pinto C, Velebit L, Shibuya K. Estimating child mortality due to diarrhoea in developing countries. Bull World Health Organ 2008;86:710-7.
Sambe-Ba B, Espié E, Faye ME, Timbiné LG, Sembene M, Gassama-Sow A. Community-acquired diarrhea among children and adults in urban settings in Senegal: Clinical, epidemiological and microbiological aspects. BMC Infect Dis 2013;13:580.
Velasco J, González F, Díaz T, Peña-Guillén J, Araque M. Profiles of enteropathogens in asymptomatic children from indigenous communities of Mérida, Venezuela. J Infect Dev Ctries 2011;5:278-85.
World Health Organization. Global Burden of Disease Estimates, 2004. Available from: http://www.who.int/healthinfo/global_burden_disease/2004_report_update_full/en.pdf. [Last update on 2007].
World Health Organization. Diarrhoea: Why Children are Still Dying and what can be Done; 2009. Available from: http://www.unicef.org/health/files/Final_Diarrhoea_Report_October_2009_final.pdf. [Last accessed on 2014 May 07].
Deen JL, von Seidlein L, Sur D, Agtini M, Lucas ME, Lopez AL, et al.
The high burden of cholera in children: Comparison of incidence from endemic areas in Asia and Africa. PLoS Negl Trop Dis 2008;2:e173.
Ghimire M. Acute diarrhoea and respiratory infections: Neglected and forgotten health problems? Com Dis Newsl 2008;5:1-2.
World Health Organization. Manual for Laboratory Investigation of Acute Enteric Infections. CDD/83.3. Geneva, Switzerland: World Health Organization; 1987.
Nguyen TV, Le Van P, Le Huy C, Gia KN, Weintraub A. Detection and characterization of diarrheagenic Escherichia coli
from young children in Hanoi, Vietnam. J Clin Microbiol 2005;43:755-60.
Kelkar SD, Zade JK. Group B rotaviruses similar to strain CAL-1, have been circulating in Western India since 1993. Epidemiol Infect 2004;132:745-9.
Gouvea V, Allen JR, Glass RI, Fang ZY, Bremont M, Cohen J, et al.
Detection of group B and C rotaviruses by polymerase chain reaction. J Clin Microbiol 1991;29:519-23.
Chhabra P, Dhongade RK, Kalrao VR, Bavdekar AR, Chitambar SD. Epidemiological, clinical, and molecular features of Norovirus
infections in western India. J Med Virol 2009;81:922-32.
Kang G, Arora R, Chitambar SD, Deshpande J, Gupte MD, Kulkarni M, et al.
Multicenter, hospital-based surveillance of Rotavirus
disease and strains among Indian children aged <5 years. J Infect Dis 2009;200 Suppl 1:S147-53.
Nair GB, Ramamurthy T, Bhattacharya MK, Krishnan T, Ganguly S, Saha DR, et al.
Emerging trends in the etiology of enteric pathogens as evidenced from an active surveillance of hospitalized diarrhoeal patients in Kolkata, India. Gut Pathog 2010;2:4.
Estrada-Garcia T, Lopez-Saucedo C, Thompson-Bonilla R, Abonce M, Lopez-Hernandez D, Santos JI, et al.
Association of diarrheagenic Escherichia coli
pathotypes with infection and diarrhea among Mexican children and association of atypical enteropathogenic E. coli
with acute diarrhea. J Clin Microbiol 2009;47:93-8.
Ochoa TJ, Ecker L, Barletta F, Mispireta ML, Gil AI, Contreras C, et al.
Age-related susceptibility to infection with diarrheagenic Escherichia coli
among infants from Periurban areas in Lima, Peru. Clin Infect Dis 2009;49:1694-702.
Dutta S, Guin S, Ghosh S, Pazhani GP, Rajendran K, Bhattacharya MK, et al.
Trends in the prevalence of diarrheagenic Escherichia coli
among hospitalized diarrheal patients in Kolkata, India. PLoS One 2013;8:e56068.
Gascón J, Vargas M, Schellenberg D, Urassa H, Casals C, Kahigwa E, et al.
Diarrhea in children under 5 years of age from Ifakara, Tanzania: A case-control study. J Clin Microbiol 2000;38:4459-62.
Checkley W, Buckley G, Gilman RH, Assis AM, Guerrant RL, Morris SS, et al.
Multi-country analysis of the effects of diarrhoea on childhood stunting. Int J Epidemiol 2008;37:816-30.
Román E, Wilhelmi I, Colomina J, Villar J, Cilleruelo ML, Nebreda V, et al.
Acute viral gastroenteritis: Proportion and clinical relevance of multiple infections in Spanish children. J Med Microbiol 2003;52:435-40.
[Table 1], [Table 2]