|Year : 2014 | Volume
| Issue : 2 | Page : 143-148
Detection of Rotavirus and Adenovirus diarrhea in children below five years, in Dakshina Kannada District, a coastal region of Karnataka State, India
Anup Kumar Shetty, Faizan Mohammad Kalekhan, Sibin Jose Muthiravalapil, Rekha Boloor, Beena Antony
Department of Microbiology, Father Muller Medical College, Kankanady, Mangalore, Dakshina Kannada, Karnataka, India
|Date of Web Publication||1-Jul-2014|
Anup Kumar Shetty
Department of Microbiology, Father Muller Medical College, Kankanady, Mangalore, Dakshina Kannada - 575 002, Karnataka
Source of Support: None, Conflict of Interest: None
Context: Diarrheal disease is the second largest cause of death of children under 5 years. Viral diarrhea is most common which is usually caused by Rotavirus followed by enteric Adenovirus. Rotaviruses are responsible for approximately 527,000 deaths mainly in low-income countries of Africa and Asia. There is limited number of studies done on viral diarrhea in this coastal belt. Aim: To determine the prevalence of Rotavirus and Adenovirus diarrhea among children below 5 years of age and its epidemiological importance in this coastal region of Karnataka State, India. The impact of factors like socio-economic status, drinking water, and Rotavirus immunization status that can contribute to the disease were also evaluated and recommendations are formulated for disease prevention. Materials and Methods: Children below 5 years of age with history of diarrhea, vomiting and fever of less than 3 days were included in this study. The sample size was 35. General physical examination, clinical findings and other details like socio-economic status, personal hygiene, growth milestones and family income were also recorded. A commercial Rotavirus and Adenovirus antigen detection kit was used to detect the presence of Rotavirus and Adenovirus antigen from the stool sample. Results: Out of 35 patients screened, 25 (71.43%) patients were negative for both Adenovirus and Rotavirus, 10 children (28.57%) were positive for Rotavirus and no patients were positive for Adenovirus. Severe dehydration, fever, loose stools and vomiting were the commonest symptoms seen in Rotavirus positive children. The highest incidence of infection with Rotavirus was seen among 7-12 months age group. Children whose stool was positive for Rotavirus used public water supply. Conclusions: Rotavirus is an important cause of diarrhea in children below 5 years in this coastal region. A simple, rapid immunochromatography test is useful and economical tool to simultaneously detect and screen Adenovirus and Rotavirus. Low socioeconomic state and unsafe water supply contributes to diarrheal disease.
Keywords: Adenovirus, children, Dakshina Kannada, diarrhea, Karnataka, Rotavirus
|How to cite this article:|
Shetty AK, Kalekhan FM, Muthiravalapil SJ, Boloor R, Antony B. Detection of Rotavirus and Adenovirus diarrhea in children below five years, in Dakshina Kannada District, a coastal region of Karnataka State, India. Muller J Med Sci Res 2014;5:143-8
|How to cite this URL:|
Shetty AK, Kalekhan FM, Muthiravalapil SJ, Boloor R, Antony B. Detection of Rotavirus and Adenovirus diarrhea in children below five years, in Dakshina Kannada District, a coastal region of Karnataka State, India. Muller J Med Sci Res [serial online] 2014 [cited 2019 Oct 16];5:143-8. Available from: http://www.mjmsr.net/text.asp?2014/5/2/143/135749
| Introduction|| |
Diarrheal disease is the second largest cause of death of children under 5 years and is responsible for 1.5 million deaths worldwide every year. World Health Organization (WHO) reports that there are two billion cases of diarrheal disease every year worldwide.  Diarrhea can be caused by bacteria, viruses, parasites, and rarely fungi. Viral diarrhea is the commonest among children under 5 years and Rotavirus being the commonest virus. Other viruses like enteric Adenovirus, Calcivirus, and Astroviruses are also known to cause diarrhea.  WHO estimated that Rotaviruses are responsible for approximately 527,000 deaths each year, with more than 85% of these deaths occurring in low-income countries in Africa and Asia, and over two million are hospitalized each year with pronounced dehydration. About 36% of hospitalizations for diarrhea among children aged <5 years were caused by Rotavirus infection. Children between 6 months and 2 years are most vulnerable to the disease. 
After Rotavirus, enteric Adenovirus has been shown to be the most common cause of paediatric viral gastroenteritis in many parts of our country.  Adenovirus can also cause infections like pneumonia, cystitis, conjunctivitis, hepatitis, myocarditis, intussusception and encephalitis apart from diarrhea. Epidemiological studies in developing and developed countries have succeeded in establishing an etiological role of enteric Adenovirus in infantile gastroenteritis in some instances, but their importance remains uncertain. , There are about 52 human Adenovirus serotypes and they are classified into six subgenera (subgenera A to F) on the basis of their biological and genetic characteristics.  Among these subgenera, subgenus F, representing Adenovirus type 40 and 41, has been found to be associated with acute gastroenteritis and is responsible for one to 20% of the cases of diarrheal disease globally in both outpatients and hospitalized children. 
There are several studies done on Rotavirus, but enteric Adenoviruses are less explored. There is limited number of studies done on viral diarrhea in this coastal belt. , This study aims to determine the prevalence of Rotavirus and Adenovirus diarrhea among children below 5 years of age and its epidemiological importance in this coastal region of Karnataka State, India. Enzyme Immunoassays (EI) and polymerase chain reaction (PCR) are commonly used for the diagnosis of Rotavirus and Adenovirus diarrhea from stool samples. , This study uses a simple and rapid immunochromatography method to detect Rotavirus and Adenovirus antigens from stool samples. The impact of factors like socioeconomic status, drinking water, and Rotavirus immunization status that can contribute the prevalence of the disease will also be evaluated and recommendations will be formulated for disease prevention.
| Materials and Methods|| |
Children below 5 years of age attending Father Muller Medical College Hospital, with history of diarrhea, vomiting and fever of less than 3 days were included in this study. The sample size was 35 and the study period was 2 months. Both out-patients and in-patients were included in the study. General physical examination and other details like socioeconomic status, personal hygiene, Rotavirus immunization history, and growth milestones were also recorded. A well-structured questionnaire which includes the personal data of the patient (name, age, sex, hospital number, Father/Mother's name, and address) was designed. History of illness such as fever, loose stools, vomiting, blood in stools, previous history of diarrhea, growth milestones, immunocompromised state and dehydration signs were also recorded. Other details such as monthly family income, methods of washing hand after using toilet were also recorded.
After verbal parental consent, about 15-20 ml of stool specimen was collected in a sterile wide mouthed container. The stool sample was transported to the laboratory immediately without any delay. The specimens were processed in Microbiology laboratory of Father Muller Medical College Hospital. A commercial Rotavirus and Adenovirus antigen detection kit (SD Bioline Rota/Adeno Rapid Test kit, Standard Diagnostics, Korea) was used to detect the presence of Rotavirus and Adenovirus antigen from the stool sample. The sensitivity and specificity of the kit were 97.4% and 100% respectively. This kit was based on immunochromatography principle and had an advantage of detecting Rotavirus and Adenovirus simultaneously with a single kit. The specimen was processed as per kit manufacturer's recommendations.
Antigen extraction from stool was done by first allowing the test device and stool sample to settle to room temperature prior to testing. An assay diluent was transferred into the sample collection tube (provided in the kit) and filled up to the marked line. A small portion of faeces (about 50 mg) was collected with the sample collection swab (provided in the kit) and it was inserted into the sample collection tube containing diluent and rolled at least 10-times properly to release the stool specimen. The swab was later discarded after squeezing it against the wall of tube.
The test device was removed from the foil pouch and placed on a flat, dry surface. A dropping cap (provided in the kit) was assembled on the sample collection tube. About 4-5 drops (about 100-125 μl) of the mixture from sample collection tube was added into the sample well of the test device. The results were interpreted within 20 minutes. There are three different dark red coloured bands that can be seen in the test device depending on the test result. Starting from the side of the sample well, the first band was T1 (Adenovirus), followed by T2 (Rotavirus) and C (Control). The presence of only control band (C) within the result window indicated negative result. The presence of two colour bands (T1 and C band) within the result window indicated Adenovirus positive. The presence of two colour bands (T2 and C) within the result window indicated Rotavirus positive [Figure 1]. The presence of three colour bands (T1, T2 and C) within the result window indicated both Adenovirus and Rotavirus positive. The approximate duration for entire procedure was 30 minutes.
| Results|| |
During the study period of 2 months 35 patients were screened and among them 25 (71.43%) patients were negative for both Adenovirus and Rotavirus, 10 children (28.57%) were positive for Rotavirus and no patients were positive for Adenovirus [Table 1]. Patients from rural area were 26 (74.29%) and from urban area 9 (25.71%). Out of the 10 children positive for Rotavirus, two (20%) were from urban area and eight (80%) from rural area. The sex distribution was 23 (65.71%) males and 12 (34.29%) females. The clinical variables of children with Adenoviral and Rotaviral diarrhea is summarized in [Table 2]. Severe dehydration, fever, loose stools and vomiting were the commonest symptoms seen in Rotavirus positive children. None of the patients had bloody diarrhea. The vital signs of the patients were within normal limits. All the 10 children who were Rotavirus positive were below 2 years of age. The highest incidence of infection with Rotavirus was seen among 7-12 months age group [Table 3]. The total monthly family income is summarized in [Table 4]. Rotavirus infections were seen in children whose monthly family income were below rupees 15,000. The source of drinking water here in this part is mostly well water and public water supply [Table 5]. Children whose stool was positive for Rotavirus used public water supply. All the members of the family washed hands after using toilet with soap and water. None of the child went to any play school or regular school. The growth milestones were uneventful for all the children. None of children were immunocompromised and no children had received Rotavirus immunization.
|Table 2: The clinical variables of children with adenoviral and rotaviral diarrhea|
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| Discussion|| |
Rotaviruses are one of the major causes of severe gastroenteritis in infants and young children worldwide.  They belong the family Reoviridae and are double stranded ribo nucleic acid (RNA) viruses. Since the first description in humans in 1973, and their subsequent recognition as a major human pathogen, a large number of studies were done on the structure, pathogenesis, and epidemiology of these viruses. Bright Singh from Pune, India reported 61 Rotavirus positive cases out of 213 cases (28.5%).  Another study reported that 266 Rotavirus cases were detected out of 945 cases (28.15%) of stool sample from children below 5 years of age with acute diarrhea.  A study done in 1993 by Desai reported 23% prevalence in hospitalized children below 5 years.  Lower prevalence rates of Rotavirus diarrhea have been reported in India from Bangalore (16.3%), Calcutta (14.6%), and Vellore (17.8%). ,, A very high higher prevalence rates in Indian studies were found in Kerala (70.7%) and Manipur (89.9%). This high prevalence of Rotavirus diarrhea was said to be because there is high rainfall, inadequate water supply, and improper night soil disposal system. , In our study Rotavirus infection was 28.57% and the results are consistent with the rates of detection of Rotaviruses among acute gastroenteritis cases in developing countries. 
Dehydration is common in Rotavirus infection and majority of them are associated with severe dehydrating diarrhea that lead to hospitalization. , Studies indicate that dehydration is seen in more than 88.5% patients. , In our study, all children with Rotavirus infection had dehydration and out of them six (80%) children had severe dehydration and had to be hospitalized [Table 2].
Children lesser than 2 years of age are vulnerable to Rotavirus infection. , In our study, six (60%) children with Rotavirus infection were in the age group of 7-12 months [Table 3]. In one study done earlier, it was suggested that infants from birth to 12 months are highly susceptible to Rotavirus and reported higher incidence of 74.1%, whereas another study reported age-specific incidence of Rotavirus diarrhea in the age group of 6-24 months which was 29.02%. , According to the WHO scientific working group, most cases of Rotavirus infection are in children between 6-24 months with a peak incidence at 9-12 months  which was observed in our study also.
In our study, out of 23 male patients, seven were positive for Rotavirus and out of 12 female patients three were positive for Rotavirus. Male incidence is up to 20% higher than that of female cases, but whether this is due to a greater susceptibility of exposure of male children or to a higher likelihood of their being brought for medical care is not known.  In our study male patients with Rotavirus positive were more than females but the data and the sample volume were not sufficient to conclude the reasons nor it was possible to comment if it was significant.
Mild fever may or may not be present and watery diarrhea is present in the case of Rotavirus infection.  In our study, out of 10 positive Rotavirus cases, nine patients (90%) had fever either at the time of examination or fever was present 2-3 days prior to hospitalization. Vomiting was the first symptom in some cases of Rotavirus infection which later progressed to acute diarrhea. All Rotavirus positive cases (100%) had diarrhea and vomiting. In the study by Singh PB 72.1% cases of Rotavirus had complaints of vomiting and diarrhea. Watery, large, frequent stools indicate severe diarrhea that requires intravenous fluid therapy. 
After Rotavirus, enteric Adenovirus has been shown to be the most common cause of paediatric viral gastroenteritis in many parts of our country.  Human Adenovirus belongs to the family Adenoviridae and known to cause acute respiratory, gastrointestinal, and urinary tract infections. They are double stranded de-oxy ribo nucleic acid (DNA) viruses. About 52 human Adenovirus serotypes have been identified and classified into six subgenera (subgenera A to F) on the basis of their biological and genetic characteristics.  Among these subgenera, subgenus F, representing Adenovirus type 40 and 41, has been found to be associated with acute gastroenteritis and is responsible for one to 20% of the cases of diarrheal disease globally in both outpatients and hospitalized children.  They are also the most prevalent serotypes associated with acute gastroenteritis in infants and children in western India.  Adenovirus can also cause infections like pneumonia, cystitis, conjunctivitis, hepatitis, myocarditis, intussusception, and encephalitis apart from diarrhea. The clinical features of Adenoviral diarrhea is characterized by watery, non-bloody diarrhea lasting for 2-22 days with mild fever and vomiting. Respiratory symptoms may accompany gastroenteritis. Göran Wadell reported that, Adenoviruses have been associated with 7-17% of cases of diarrhea in children and concluded that the impact of enteric Adenoviruses in the aetiology of diarrhea world-wide is not known, but is accessible to investigation.  In Japan, the high incidence (8%) of Adenovirus infection made Adenovirus a second common agent of acute gastroenteritis.  There are very few studies done on the prevalence of Adenovirus diarrhea in this belt of coastal region. One study done in this coastal belt showed very less prevalence of Adenovirus (3%).  In our study all the analyzed stool samples were negative for Adenovirus infection and are contrary to the rates reported in other parts of our country.  However this does not rule out complete absence of Adenoviral diarrhea in this region if not lower prevalence. A larger sample size with longer study period would have given a better picture of the prevalence.
Enzyme immunoassays are sensitive for the diagnosis of Rotavirus and Adenovirus diarrhea from stool samples. The rapid immunochromatography commercial kit used in this study had a sensitivity and specificity of 97.4% and 100% respectively. It would be difficult to comment if more cases would have been detected if better methods like PCR or Enzyme linked Immunosorbent assay (ELISA) were used in screening since we have not compared with these methods. Screening the samples with PCR or ELISA have better sensitivity than immunochromatography but would be costlier and not practical. The rapid immunochromatography commercial kit used in this study for detection of Rotavirus and Adenovirus is economical and practical. It can also be used at the bedside and even in field studies. However PCR is recommended for detection in immunocompromised patients. ,
Prevalence of viral diarrhea is higher in low socioeconomic state and in children using public water supply, which is observed universally across the globe, particularly in developing countries. The findings of this study were consistent with global findings. A Nigerian study showed that greater proportions of the Adenovirus-positive cases were aged between 13-24 months and there was no difference in the prevalence of the infection between male and female. However, the source of drinking water had a significant effect on the frequency of stool per day. 
Vaccines offer the most promising tool for preventing morbidity and mortality caused by Rotavirus. Licensed and safer Rotavirus vaccine is available currently and effective in preventing the disease.  An outbreak of epidemic gastroenteritis in a Portuguese municipality showed an overall attack rate was 11·4% with Adenovirus diarrhea. This high infection rate of Adenovirus was due to high vaccination coverage against Rotaviruses in children aged <2 years, thus proving that Rotavirus vaccination is effective when included in vaccination schedule.  The epidemiological profile of these viruses in India will be of considerable importance to both policy makers and vaccine developers in determining the composition, dosage, and schedule for a vaccine to be used in India. However improvement of socioeconomic state, safe drinking water and patient education will have a significant impact on preventing Rotavirus & Adenovirus diarrhea.
| Conclusion|| |
Rotavirus is an important cause of diarrhea in children below 5 years, particularly in children below 2 years in this coastal region. Studies with larger sample size are required to determine the true prevalence of Rotavirus and Adenovirus diarrhea in this region. A simple, rapid immunochromatography test is useful and economical tool to simultaneously detect and screen Adenovirus and Rotavirus from stool samples of children. Low socioeconomic state, unsafe water supply contributes to occurrence of viral diarrhea. Providing safe drinking water is the most important factor in preventing the disease. Routine immunization with Rotavirus vaccine in this region may be considered to aid prevention.
| Acknowledgement|| |
Partly sponsored by Indian council of medical research (ICMR) under the Short term studentship (STS) programme.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]