Mansabdar, Sambrani, and Kumar S: A study of rotavirus infection in acute diarrhoea in children less than 5 years of age


Introduction

Diarrheal diseases account for majority of deaths globally making it the second leading cause of death in childhood.1 In India diarrhea accounts to 14% deaths in children less than 5 years of age.2

Diarrhea could be due to various causes like viruses, bacteria, protozoa and helminths. In developing countriesb50-60% cases are bacterial in origin were Escherichia coli amounts to 25%, Campylobacter jejuni 10-15%, Salmonella species 5%, Shigella species 5%, followed by 35% viral etiology, were rotavirus accounts for 15-25% and in many the cause is unidentified or mixed.3

In India, acute diarrheal disease accounts to 8% deaths in children under 5 years age. During 2013, about 10.7 million cases with 1535 deaths have been reported in India.4 Although acute diarrheal disease is usually self limiting, infectious source needs proper work up and treatment.

Rotavirus infection is most common amongst the viral etiology leading to 20-30% hospitalized cases in India under 5 years age and estimated to cause approximately 5,27,000 deaths each year.5

Hence the present study was taken up for rapid diagnosis of rotaviral infections, to prevent the irrational use of antibiotic therapy and prolonged hospitalization.

Materials and Methods

The present study was a cross sectional, prospective study conducted at the department of Microbiology, from December 2015 to November 2016.

Source of data

Stool samples from patients admitted in the paediatric ward of were received at Department of Microbiology.

Sample size

Total of 100 cases were included in the study.

Inclusion criteria

  1. All children below 5 years of age presenting with acute diarrhoea and hospitalized for same, were included in the study.

  2. All children enrolled were not vaccinated for rotavirus.

Exclusion criteria

  1. Diarrhoea for more than 14 days / persistent diarrhoea.

  2. Children with HIV positive status.

  3. Children with dysentery.

  4. Outdoor patient

  5. Patient not giving consent

Methodology

A detailed clinical history was taken, thorough clinical examination was done and duly recorded in the case record forms. An evaluation of degree of hydration was done as per WHO criteria.

Subsequently stool sample was sent to department of microbiology for ELISA for Rotavirus detection.

  1. KIT used: Premiere Rotaclone, Meridian Biosciences, Inc

  2. LOT number: 6042.074

  3. Expiry date: 2017-02-04

Principle

Solid phase sandwich type EIA. Plastic microtitre wells are coated with a monoclonal antibody directed against the product of the sixth viral gene (VP6), which is the group specific antigen for all known human rotaviruses.

Procedure

  1. Specimen preparation- Add 1 ml of sample diluent to a marked tube, using a transfer pipette.

  2. Snap off sufficient number of wells for samples and the controls. Insert into the microtitre well holder.

  3. Add 2 drops(100 µl) each of diluted fecal sample, positive and negative control to the bottom of separate wells.

  4. Add 2 drops(100µl) of enzyme conjugate to each well. Mix by gently swirling on the table top.

  5. Incubate at room temperature for 60 minutes.

  6. Pour the liquid out of the wells into a discard jar. Tap the microtitre plate holder upside down vigorously against the absorbent paper to ensure complete removal of liquid from the wells.

  7. Fill all the wells to overflow with deionised water and pour the liquid out as in step 5

  8. Repeat the washing procedure four times (a total of 5 washes)

  9. Add 2 drops (100 µl) of substrate solution to each well

  10. Add 2 drops(100µl) of chromogen solution to each well

  11. Incubate 10 minutes at room temperature

Spectrophotometric procedure :

This can be determined by adding 2 drops of stop solution (sulphuric acid) to each well.

Interpretation of result

  1. Positive results by visual determination: Any sample with blue colour more than intense than that of negative control is considered positive.

  2. Positive results by spectrophotometric determination: Specimens with absorbance unit A(450) greater than 0.150 are considered positive.

Statistical analysis of data was done using IBM SPSS statistical software version 22.

Ethical consideration

The ethical clearance of the study has been obtained from the instituitional scientific committee.

Results

A total of 100 cases of acute diarrhoea in children less than 5 years, who were admitted in paediatric department, KIMS Hospital, Hubballi, were studied during the December 2015 to December 2016.

Table 1

Showing age and gender distribution of diarrhoeal cases and its comparison to rotavirus positive cases

Age in months

Male

Female

Rotavirus positive

Less than 6

06

04

02(6.89%)

6-12

24

24

16(55.17%)

1-2 years

17

09

09(31%)

>2-5years

07

09

02(6.89%)

Total

54

46

29

Observation

Maximum rotavirus positive cases were seen in the age group of 6-12 months (55.17%), followed by 1-2 years (31%) and least were seen among less than 6 months and 2-5 years. Also rotavirus positive cases in males and females shows 62.06% and 37.93% respectively.

Table 2

Showing the urban - rural distribution of rotavirus cases

Place

No. of. cases of Rotavirus

Non Rotavirus

Urban

10(34.48%)

20

Rural

19 (65.5%)

51

The chi-square statistic is 0.3909. The p-value is .531852. This result is not significant at p<0.05.

Observation

The Table 2 shows that rural rotavirus positive cases are 65.5% when compared to urban population which shows 34.48%.

Graph 1

Showing seasonal distribution of rotavirus infection

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Observation

Above Table 2 that majority of the rotavirus positive cases were seen in the winter season which accounted to 85.37%, 10% in rainy season and 3.44% in summer season.

Graph 2

Diet and rotavirus correlation

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Observation

Above Table 2 that maximum number of cases (75%) shows rotavirus positive when the children lack exclusive breast feeding whereas 24.13% in children who were exclusively breast fed.

Table 3

Showing correlation of frequency of loose stools with rotavirus positive cases

No. of stools/ day

Rotavirus positive

Rotavirus negative

<=5

05(17.24%)

21

>5<10

08(27.58%)

24

=>10

16(55.17%)

26

Observation

The Table 3 shows that samples having history of passing >10 stools per day had 55.17% of rotavirus positivity, followed by 27.58% when stools are <10 in number and 17.24% when it is less than 5 stools/day

Table 4

Showing correlation of associated complaints with rotavirus positive cases:

Complaints

No. of cases with rotavirus

Non rotavirus cases

Only diarrhoea

05(17.24%)

19

Diarrhoea with fever

06(20.6%)

17

Diarrhoea with vomiting

03(10.34%)

18

Diarrhoea with vomiting and fever

15(51.72%)

17

Observation

Shows that almost 51.72% cases of rotavirus had complaints of diarrhoea, fever and vomiting, followed by 20.6% with diarrhoea and fever, 17% with only diarrhoea and 10.34% with diarrhoea and vomiting.

Graph 3

Showing assessment of dehydration with rotavirus positive cases

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Observation

Maximum number of cases of rotavirus positive samples were showing some dehydration(58.62%), followed by equal distribution of cases in no dehydration and severe dehydration which had 17, 6 and 6 respectively. Among severe dehydration Rotavirus cases were 66.66%.

Table 5

Showing stool macroscopy in acute diarrhoea cases

Type

No. of Cases

Solid stools

08

Yellowish stool

70

Greenish stools

22

Observation

Above Table 5 that 70% cases were presenting with yellowish coloured stools, followed by 22% showing greenish stools and 8% with solid stools1showing macroscopic picture of greenish frothy stools with mucous flakes of rotavirus positive specimen:

Figure 1

Showing macroscopic picture of greenish frothy stools with mucous flakes of rotavirus positive specimen

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Figure 2

Showing ELISA plate: visual determination of rotavirus antigen detection Blue colour well: positive, colourless well: negative

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Figure 3

ELISA plate showing rotavirus positive after adding STOP solution: spectrophotometric determination: yellow colour shows Positive for Rotavirus antigen

https://typeset-prod-media-server.s3.amazonaws.com/article_uploads/f2666012-b2c7-4afd-afc4-d16bea15f61f/image/320ddf90-ac33-49ee-9959-8cca766a58a2-uimage.png

Discussion

This study was conducted in the department of microbiology from December 2015- December 2016.

The total number of acute diarrhoeal cases admitted to paediatric ward during this period were 186 in accounting to prevalence rate of 5.86% in our set up.

Among them, 100 cases were included in the present study for acute diarrhoea which were within our inclusion criteria.

In the present study maximum rotavirus positive cases were seen in the age group of 6-12 months (55.17%), followed by 1-2 years(31%) and least were seen among less than 6 months and 2-5 years. Also rotavirus positive cases in males and females shows 62.06% and 37.93% respectively. Also in a study conducted by Satarupa Mullick et al 6 in 2014 most cases of ADD belonged to 6months to 2 years, followed by 0-6months of age. In another study done by Shaveta Dhiman et al 7 in 2015 at Amritsar maximum cases of ADD (69%) belonged to 6 months - 2 years of age. These findings were similar to the present study. Predominant number of cases in the age group of 6-12 months may be due to the commencement of weaning period, which leads to exposure to the water and other contaminants and also during this period infant starts toddling and acquire the habit of mouthing. 8

Table 6

Showing commonly affected age group due to rotavirus infection

S.No

Author

Year

Place

Total no. of study cases

Commonly involved age group

1

Shaveta Dhiman et al 7

2015

Amritsar

100

6months-2 years

2

Anup kumar Shetty et al 9

2014

Karnataka

35

7months- 2 years

3

Ma Mathew et al 10

2013

Kerala

1827

6 months to 2 years

4

Muhammed Irfan Habib et al11

2007

Karachi

300

6months-2 years

5

Pierre Van Damme et al12

2005

Europe

2846

6months-2 years

6

Present study

2016

Hubballi

100

6months-2 years

In the present study out of 100 children presenting with acute diarrhoea 54% were males and females accounted for 46%. In a study at Tumkur, Karnataka, from Nov 2007 –Aug 2009, the prevalence of diarrhea in children accounted for 10.12%. Out of 150 cases enrolled, maximum cases of diarrhea occurred in the first 24 months of life 107 (71.3%), followed by 24-60 months (15.3%) and 6-12 years (13.3%).13 In a study conducted by Manohar Badur et al in 2015 in Tirupati, predominant number of males were affected which is around 57.2% and females were 42.8%.14 Thus the results are almost similar to the present study. The rotavirus cases in present study accounted for a total of 29%, with a predominant male pattern with 62.06% and females showing 37.93%, showing ratio of 1.5:1. In a study conducted at Lucknow during August 2010-July 2012, rotavirus was the most frequent cause of viral gastroenteritis in both hospitalized and non-hospitalized children accounting for 19.5% and 9.1% respectively, 15 Rajiv Bahl et al16 in 2005 at New Delhi showed 26% positivity, Satarupa Mullick et al6 in 2014 at Kolkata showed 25.2%, Manohar Badur et al 14 in 2015 at Tirupati showed 25.67%. In a study conducted by Shobha Ram et al(1990) at Chandigarh observed M:F of 3.3:1, for rotavirus infection. 17 The present study correlates with study by P. Sarvanan et al(2004), 18 Pune, 19 Karachi20 and Turkey21 which detected rotavirus antigen in equal frequency in both males and females.

Seasonwise distribution of Rotavirus diarrhoea:

In the present study 88% cases were detected in the winter, 3.44% in summer and 10% during rainy season.

In a study conducted in southern Orissa from Jan 2007-Dec 2010 among children less than 5 years of age; higher prevalence of diarrhoea was noticed in July to October that is during monsoon period.22

M.R. Saha et al (1984) at Calcutta detected frequent rotavirus infection during cooler months of the year.23 A study at Nepal by M. Shariff et al(2003) observed that rotavirus infections occurred throughout the year.24

A study conducted at Vietnam reported that rotavirus infection occuredall round the year, with a peak in winter season.25

A study demonstrated that the degree of sensitivity and specificity of LA test showed a roughly linear relationship with the degree of ELISA sensitivity and specificity, and the positive predictive value of the test was high.26 According to Raj et al, the LA kit studied was a specific and rapid method. While it may be useful in certain situations, such as in outbreaks, its low sensitivity can make it unsuitable for use in routine clinical practice.27

Conclusion

Incidence of rotavirus cases in and around Hubballi is 29% in children less than 5 years of age. The study shows that maximum rotavirus cases were seen in the age group of 6-24 months. As the samples collected were from unvaccinated children, it also relates the importance of vaccination which could prevent dehydration resulting from rotavirus infection.Also, a rapid diagnosis of these infections with EIA, may be valuable in the formulation of prognosis for children with acute diarrhoea and may prevent the irrational use of antibiotic therapy and prolonged hospitalization.

Source of Funding

None.

Conflict of Interest

The authors declare no conflict of interest.

References

1 

B Ahmed RM Kliegman BF Stanton JW StGeme NF Scholr RE Behrman Acute gastroenteritis in childrenNelson textbook of paediatrics19th editionWB Saunders CoPhiladelphia201113239

2 

U Isalkar Times of India 50 pc medical staff dump ORS28September2010

3 

S Ansari SB Sherchand K Parajuli SK Mishra RK Dahal S Shrestha Bacterial etiology of acute diarrhea in children under five years of ageJ Nepal Health Res Counc2012102221823

4 

Park textbook of preventive and social medicine23rd editionJP Publication2015221

5 

N Jindal S Arora R Arora Enterotoxigenic Enteric bacteria causing secretory DiarrhoeaIndian J Pathol Microbiol199538217780

6 

S Mullick A Mukherjee S Ghosh GP Pazhani D Sur B Manna Community based case-control study of rotavirus gastroenteritis among young children during 2008-2010 reveals vast genetic diversity and increased prevalence of G9 strains in KolkataPLoS One2014911e112970

7 

S Dhiman B Devi K Singh P Devi Comparison of Enyme Linked Immunosorbent Assay and Immunochromatography for Rotavirus Detection in children below five years with acute gastroenteritisJ Clin Diagn Res20159916

8 

RC Pande VS Rajvanshi SC Gupta TN Mehrotra Enteropathogenic serotypes of Escherichia coli isolated from sporadic cases of acute diarrhoea and Gastroenteritis and chronic Diarrhoea in AllahabadIndian J Med Res19736111158893

9 

PV Damme C Giaquinto M Maxwell P Todd Distribution of Rotavirus Genotypes in Europe, 2004–2005: The REVEAL StudyJ Infect Dis20071951S172510.1086/516715

10 

MA Mathew A Paulose S Chitralekha MK Nair G Kang P Kilgore Prevalence of Rotavirus Diarrhea among Hospitalized Under-five ChildrenIndian Pediatr2014512731

11 

AK Shetty FM Kalekhan SJ Muthiravalapil R Boloor B Antony Detection of Rotavirus and Adenovirus diarrhea in children below five years, in Dakshina Kannada District, a coastal region of Karnataka State, IndiaMuller J Med Sci Res201451438

12 

MI Habib SG Kazi KA Khan N Zia Rota virus diarrhea in hospitalized childrenJ Coll Physicians Surg Pak20142421147

13 

KR Haricharan BM Shrinivasa Vatsalakumari Clinical and Bacteriological Study of Acute Diarrhoea in ChildrenJ Evol Med Dent Sci2013223422937

14 

M Badur NM Latha PR Kumar SR Dudala SA Shaik G Kang Prevalence of rotavirus diarrhoea among under 5 hospitalized children in a government tertiary hospitalJNTR Univ Heal Sci2015421126

15 

S Gupta KP Singh A Jain S Srivastava V Kumar M Singh Aetiology of childhood viral gastro-enteritis in Lucknow, north IndianIndian J Med Res201514146972

16 

U Ganguly D Sen P Dutta SC Pal Enterotoxigenix Escherichia coli associated diarrhoea in children and adults at a hospital in CalcuttaIndian J Med Res198071498500

17 

A Borade AS Bais V Bapat R Dhongade Characteristics of rotavirus gastroenteritis in hospitalized children in PuneIndian J Med Sci201064252108

18 

P Saravanan S Ananthan M Ananthasubramanian Rotavirus infection among infants and young children in Chennai, south IndiaIndian J Med Microbiol200422421221

19 

R Bahl P Ray S Subodh P Shambharkar M Saxena U Parashar Incidence of severe rotavirus diarrhea in New Delhi, India, and G and P types of the infecting rotavirus strainsJ Infect Dis2005192Supplement_1S114

20 

R Durmaz AT Kalaycioglu S Acar Z Bakkaloglu A Karagoz G Korukluoglu Prevalence of Rotavirus Genotypes in Children Younger than 5 years of age before the introduction of a Universal Rotavirus vaccination Program: Report of rotavirus Surviellance in TurkeyPLoS One2014912e113674

21 

Rotavirus and other viral diarrhoeas, WHO scientific working groupBull World Heal Organ198058218398

22 

MV Narsimham SK Sahu P Panda B Parida Incidence of bacterial enteropathogens among hospitalised children less than 5 years with Diarrhoea in southern OrissaIndian Med Gazette20143049

23 

MR Saha D Sen P Datta D Datta SC Pal Role of rotavirus as the cause of acute pediatric diarrhoea in CalcuttaTrans R Soc Trop Med Hyg198478681820

24 

AZ Kapikian J Flores Y Hoshino RI Glass K Midthun, M Gorziglia The Major etiologic agent of severe infantile diarrhoea may be controllable by aJ Infect Dis1986153581522

25 

TV Nguyen PL Van CL Huy A Weintaub Diarrhoea caused by rotavirus in children less than 5 years of age in Hanoi, VietnamJ Clin Microbiol20044212574550

26 

YAR AL- Khafaji HJ AL-Jiboury Detection of Rotavirus in diarrhea stool samples of children with acute gastroenteritis in Babylon governorate IraqInt Res J Microbiol201343848

27 

P Raj MK Bhan AK Prasad R Kumar S Sazawal N Bhandari Evaluation of a new latex agglutination kit for detection of human rotavirus in fecal specimensIndian J Med Res1989891659



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Received : 22-10-2021

Accepted : 06-12-2021

Available online : 15-12-2021


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