SYNOPSIS
Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore
“DETECTION OF ADENOVIRAL DIARRHOEA IN CHILDREN BELOW FIVE YEARS, IN RURAL AND URBAN AREAS OF DAKSHINA KANNADA DISTRICT, KARNATAKA STATE.”
Name of the candidate : Mr. SIBIN JOSE. M
Guide : Dr. ANUP KUMAR SHETTY.
Course and Subject : M.Sc. MEDICAL LABORATORY
TECHNOLOGY (MICROBIOLOGY)
DEPARTMENT OF MICROBIOLOGY
FR. MULLER MEDICAL COLLEGE HOSPITAL
KANKANADY, MANGALORE – 575 002
1 / Name of the Candidate and Address / Mr. SIBIN JOSE. MM Sc. MEDICAL LABORATORY TECHNOLOGY
(MICROBIOLOGY)
FATHER MULER MEDICAL COLLEGE, MANGLORE-575002
2 / Name of the Institution / FATHER MULLER MEDICAL COLLEGE
KANKANADY, MANGLORE- 575002
3 / Course of study and subject / M.Sc. MEDICAL LABORATORY TECHNOLOGY(MICROBIOLOGY)
4 / Date of admission to Course / 30/09/2011
5 / TITLE OF THE TOPIC:
“DETECTION OF ADENOVIRAL DIARRHOEA IN CHILDREN BELOW FIVE YEARS, IN RURAL AND URBAN AREAS OF DAKSHINA KANNADA DISTRICT, KARNATAKA STATE.”
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE
ANNEXURE –II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR PROJECT
6. / BRIEF RESUME OF THE INTENDED WORK6.1 NEED FOR THE STUDY
Diarrhoeal disease is the second largest cause of death of children under five years and is responsible for 1.5 million deaths worldwide every year. World health organization (WHO) reports that there are two billion cases of diarrhoeal disease every year worldwide. (1) Diarhhoea can be caused by bacteria, viruses and parasites. Viral diarrhoea is the commonest among children under five years and Rotavirus is the commonest virus. Other viruses like enteric Adenovirus, Calcivirus, Astroviruses are also known to cause diarrhoea. (2)
Human adenovirus belongs to the family Adenoviridae and known to cause acute respiratory, gastrointestinal, and urinary tract infections. 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 (3). Among these subgenera, subgenus F (AdV F), representing adenovirus type 40 (AdV-40) and AdV-41, has been found to be associated with acute gastroenteritis and is responsible for 1- 20% of the cases of diarrheal disease globally in both outpatients and hospitalized children. (4)
There are several studies done on Rotavirus, but enteric Adenoviruses are less explored. There is limited number of studies done on Adenoviral diarrhoea in this belt and across the country. (2,5). This study aims to determine the prevalence of Adenoviral diarrhoea among children below five years of age and its importance epidemiologically. The impact of other factors like socio-economic status, hygiene, immunization status will also be evaluated.
6.2 REVIEW OF LITERATURE
Adenovirus is associated with various diseases including respiratory, ocular & gastro intestinal infections. It’s the 2nd most important cause of acute infantile gastro enteritis after rotavirus. Most frequently associated with gastro enteritis are sub groups A (type 12, 18, 37) & F (types 40, 41). So A & F referred as enteric adenoviruses. There is no difference in prevalence of infection between male & female. Enteric adenoviral diarrhea is often protracted, but vomiting & fever are less prominent than with rotavirus. In electron microscopy 80 nm viral particle in stool can be identified. (6)
As per the data obtained from the Census India , 16.5 % of children below the age of six suffer from the diarrhoeal diseases in rural areas. While 12.4% of children of the same age group suffer in urban areas. 83.5% of children above the age of six suffers from diarrhoeal disease in rural areas, while in urban areas about 87.3% of the same age group suffers from diarrhoea.(7)
Human adenovirus belongs to the family Adenoviridae and known to cause acute respiratory, gastrointestinal, and urinary tract infections. 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 (3). Among these subgenera, subgenus F (AdV F), representing adenovirus type 40 (AdV-40) and AdV-41, has been found to be associated with acute gastroenteritis and is responsible for 1- 20% of the cases of diarrheal disease globally in both outpatients and hospitalized children. (4) Enteric adenoviral infection causes mild- moderate dehydration. It is identified that Ad 41 is more common than Ad 40.(8) Enzyme linked immunosorbent assay (ELISA) can be used for detection of Antiviral antigen. Complement fixation tests (CFT), Haemagglutination (HI), ELISA etc are used to detect antiviral antibody. There is no significant difference between the Antiviral groups with respect to results such as ESR, peripheral leucocyte count & serum electrophoresis. Radio-immunoassay (RIA) & ELISA are some recent methods for detection of Adenovirus.(9)
A commercial latex agglutination test for diagnosis of adenovirus in diarrhoeal disease was evaluated by comparison with the results obtained by ELISA, electron microscopy and virus isolation. The latex agglutination test was found to be a rapid and simple method for the detection of adenovirus in diarrhoeal disease. Compared to ELISA and electron microscopy, the sensitivity was 100% and 95% respectively and the specificity 100%.(16)
Two ELISA techniques can be used for the detection of Adenovirus in stool. One is group specific which detects enteric adenoviruses associated with infantile gastroenteritis second is type specific which selectively detects Enteric adenovirus.(10) Adenovirus serologically related to types 11,17,32 33 were infectious but non-cultivable because of its incapability to replicate in conventional cell cultures.(11) Dot- blot hybridization allows detection of fastidious enteric adenovirus & simultaneous typing viruses as Ad 40 or Ad 41.Cloned Pst1 fragments of Ad 40& Ad 41 were used as P –labelled probes in the test, which allows detection of picogram quantities of viral DNA.(12) In 60-80% of symptomatic cases adenovirus detected by Electron Microscopy is enteric type. Adenoviral gastro enteritis can be seen as co- infection with Salmonella serotypes, Enteropathogenic Escherichia coli, Campylobacter jejuni, Giardia lamblia etc. In tropical climate the incidence of enteric adenovirus was less than that found in areas with temperate climate.(13)
PCR has found to be fast, sensitive & reliable method for the detection of Adenovirus in diarrhoeal disease, if amplifications are performed directly on diluted stool sample. General primers can be used whenever fastidious infections are suspected. Selective primers are used for detecting Adenovirus directly in a sample.(14) Maternal education is important for the prevention of Adenoviral gastro enteritis. The risk factors for mortality are lack of breast feeding, recent measles, very low household income etc.(15)
6.3 OBJECTIVES OF THE STUDY
a) To determine the prevalence of Adenoviral diarrhoea in children below five years.
b) To evaluate if socio-economic status, hygiene, immunization etc can contribute the prevalence of the disease.
c) To formulate recommendations for disease prevention.
7.MATERIALS AND METHODS
7.1 SOURCE OF DATA
Sample size: 50
Out-patients and in-patients from rural and urban areas of Dakshina Kannada district, attending father Muller Medical college hospital, with history of diarrhoea, vomiting and fever of less than three days will be included in this study.
7.2 METHOD OF COLLECTION OF DATA
Inclusion criteria:
1. Children below five years, both males and females.
2. Out-patients and in-patients attending father Muller Medical college hospital, with history of diarrhoea (more than three episodes of loose stools per day), vomiting and fever of less than three day.
Exclusion criteria:
1. Children above five years.
2. Diarrhoea, vomiting, fever and other gastroenteritis symptoms of more than three days duration.
Apart from physical examination, other details like socio-economic status, personal and environmental hygiene, immunization history, growth milestones will also be recorded.
Specimen processing:
After consent, the stool specimen will be collected in a sterile wide mouthed container. About 15-20ml of stool sample is collected. The stool sample is transported to the laboratory immediately without any delay. If delay is anticipated the specimens are stored at 2-80c in a refrigerator for maximum overnight.
The specimen will be processed in Microbiology laboratory. A commercial adenovirus detection kit, HiAdenovirus Latex test kit (HiMedia laboratories, Mumbai) will be used to detect the presence of Adenovirus antigen from the stool sample. The sensitivity and specificity of the kit are 96% and 99% respectively. The specimen will be further processed as per kit manufacturer’s recommendations.
· 100µl of sample will be diluted with 1.0ml of sample diluent, in a sterile screw capped 15ml centrifuge tube. The mixture is kept at room temperature for 2 mins.
· The tube is centrifuged for 10 minutes in a centrifuge at 1000g.
· 50µl of the supernatant is taken on the test slide provided in the kit. To this one drop of adenovirus latex reagent will be added using a dropper provided in the kit.
· The mixture is mixed well with a stick provided in the kit and checked for any visible agglutination within two minutes.
Clear visible agglutination is considered positive. No visible agglutination is considered negative. Any agglutination above two minutes will be considered negative.
7.3 Does the study require any investigations or interventions to be conducted on patients or humans or animals? If so, please describe briefly.
Yes. Stool sample of children below five years with history of diarrhoea of less than three days will be subjected to Latex agglutination test for detection of Adenoviral antigen.
7.4 Has ethical clearance been obtained from your institution in case of 7.3
Yes
8. LIST OF REFERENCES
1. Accessed from internet http://www.who.int/mediacentre/factsheets/fs330/en/index.html
2. Dey RS, Ghosh S, Sarkar MC, Panchalingam S, Natro JP et.al. Circulation of a novel pattern of infections by enteric Adenovirus Serotype 41among children below five years of age in Kolkata, India. J. Clin. Microbiol:49(2),500-5.
3. Wadell, G. 1984. Molecular epidemiology of human adenoviruses. Curr. Top. Microbiol. Immunol. 110:191–220.
4. Li, L., et al. 2004. Characterization of adenovirus type 41 isolates fromvchildren with acute gastroenteritis in Japan, Vietnam, and Korea. J. Clin. Microbiol. 42:4032–9.
5. Shetty M, Brown TA, Kotian M, Shivananda PG. Viral diarrhoea in rural coastal region of Karnataka, India. J Trop Pediatr:41(5):301-3.
6. Elliott. EJ. Viral diarrhoeas in childhood, British medical journal 1992; vol: 305:
1111-1112.
7. Accessed from internet – URL: www.census india.net, Census of India, 2001.
8. Khan. KJ, Tzipori SR, Unicomb. LE. Enteric Adenoviral infection among infants with diarrhea in rural Bangladesh, Journal of clinical Microbiology 1993; 31(3) : 484 - 489.
9. Unhoo. I, Wadell. G , Svensson. L , Johansson ME. Importance of enteric Adenoviruses 40 & 41in acute gastroenteritis in infants and young children, Journal of clinical microbiology 1984 ; 20(3): 365 –372.
10. Johansson. ME, Unhoo. I, Kidd. AH, Madeley. CR & Wadell. G. Direct identification of Enteric Adenovirus, a candidate new serotype associated with Infantile Gastroenteritis, Journal of clinical microbiology 1980; 12(1) : 95 – 100.
11. Gary. GW, Hierholzer. JC & Black. RE. Characteristics of non cultivable Adenoviruses
associated with diarrhoea in infants : A new subgroup of human Adenoviruses. Journal of clinical microbiology 1979 ; 10(1) : 96- 103.
12. Kidd. AH, Harley. AH & Erasmus. MJ, Specific detection & typing of Adenovirus Types 40 and 41in stool specimens by Dot – Blot Hybridization, Journal of clinical microbiology 1985; 22(6) : 934- 939.
13. Herrmann. JE, Blacklow. NR, Henry. DM, Clements. E, Taylor. PN, Incidence of Enteric Adenoviruses among children in Thailand and the significance of these viruses in gastroenteritis, Journal of clinical microbiology 1988 ; 26(9) : 1783 – 1786.
14. Allard. A, Girones. R, Juto. P, Wadell. G. Polymerase chain reaction for detection of Adenoviruses in stool samples. Journal of clinical microbiology 1990 ; 28(12):
2659 – 2667.
15. Cook. GC. Diarrhoeal disease : a world wide problem. Journal of the royal society of medicine 1998 ; 91: 192 – 194.
16. Grandian. M, Petterson. CA, Svensson. L, Uhnoo. I. Latex agglutination test for adenovirus diagnosis in diarrhoeal disease, Jmed virol 1987 ; 23(4) : 311 – 316.
9. / SIGNATURE OF THE CANDIDATE:
10. / REMARK OF THE GUIDE: / 1. This study will determine the prevalence of Adenoviral diarrhoea in children below five years.
2. Depending on the results obtained recommendations will be formulated to prevent the occurrence of Adenoviral infections.
3. A need for routine screening of Adenoviral diarrhoea for suspected viral diarrhoea cases will also be evaluated.
11. / NAME AND DESIGNATION OF (in block letters)
11.1 GUIDE / Dr. ANUP KUMAR SHETTY Associate Professor
Department Of Microbiology
Father Muller Medical
College, Kankanady
Mangalore – 575002
11.2 SIGNATURE
11.3 HEAD OF THE
DEPARTMENT / Dr. B. REKHA
Professor
Department Of Microbiology
Father Muller Medical College, Kankanady
Mangalore - 575002
11.4 SIGNATURE
12. / 12.1 REMARKS OF THE CHAIRMAN AND DEAN
12.2 SIGNATURE